http://2007.igem.org/wiki/index.php?title=Special:Contributions/Jmonk&feed=atom&limit=50&target=Jmonk&year=&month=2007.igem.org - User contributions [en]2024-03-29T02:39:26ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-27T03:17:28Z<p>Jmonk: /* Bio-Brick Verification: Quality Control */</p>
<hr />
<div><html><br />
<img src="http://img341.imageshack.us/img341/8195/logo5oz0.jpg" alt="LOGO"><br />
</html> [[Princeton/overview | Overview]] || [[Princeton/People | People ]] || [[Princeton/lab | Lab work]] || [[Princeton/lab/experimentation | Experiments]] || [[Princeton/lab/bioinformatics | Bioinformatics]] || [[Princeton/lab/simulation | Simulations]]<br />
=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
=Assembly Line Flow=<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
[[PCR]]<br />
<br />
[[Run Gel]]<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction<br />
<br />
[[Digestion with Restriction Enzymes]]<br />
<br />
[[CIP Treatment]]<br />
<br />
[[PCR purification]]<br />
<br />
[[Ligation]]<br />
<br />
[[Transform and pick colonies]]<br />
<br />
[[Miniprep and determine concentrations]]<br />
<br />
[[Restriction Map]]<br />
<br />
[[Retransform with chosen plasmid]]<br />
<br />
[[Maxiprep and determine concentraions]]<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
Please see [[Making A Lentivirus]] for protocols<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS<br />
<br />
Please see [[Making A Lentivirus]] for protocols</div>Jmonkhttp://2007.igem.org/wiki/index.php/Making_A_LentivirusMaking A Lentivirus2007-10-27T03:16:30Z<p>Jmonk: </p>
<hr />
<div>Making a Lentivirus<br />
<br />
We used 293 FT Cells (Human Embryonic Kidney cells). Get them from the -80 C freezer (short term storage) or liquid nitrogen (long term storage).<br />
1. Warm cells in a 37 C bath, thaw until only a tiny chunk of frozen material is left. <br />
2. Add cells to 5 mL of medium.<br />
3. Spin down cells once you put them in the 5 mL medium for 5 minutes at 300 rcf.<br />
4. From spun down cells using Pasteur pipette suck out supernatant, add 700 ul of medium, resuspend.<br />
5. Spread over a 15 cm dish with 25 mL medium or 10 cm dish with 10 mL medium and swirl carefully.<br />
<br />
• Incubator = 37 C and 5% CO2 and water at bottom.<br />
• Anything going into hood gets sprayed with 70% EtOH<br />
<br />
Culture/Growth Medium:<br />
0.85 liter of DMEM (from Invitrogen)<br />
100 mL of serum (all purpose fetal calf serum; not ES cell qualified serum!)<br />
10 mL of 100mM sodium pyruvate (from GIBCO)<br />
10 mL of 100x MEM NEAA (non-essential amino acids from GIBCO)<br />
10 mL of 200mM L-glutamine (from GIBCO)<br />
10 mL of 100x 2-Mercaptoethanol (From SIGMA)<br />
(To make 100x stock, add 37 uL of Mercapto Ethanol = 14.2 M, to 100 mL of PBS)<br />
10 mL of Penicillin-Streptomycin 100X (from GIBCO)<br />
<br />
Splitting Cells<br />
<br />
1:4 split (or 1:5 depending on cell density)<br />
<br />
1. 25 mL medium for each of 4 (or 5) 15cm dishes<br />
2. Remove medium from first dish (dish with cells) with Pasteur pipette<br />
3. Add 2ml of Trypsin (brand: TrypLE Express) to first dish, swirl around and try to get all the cells off<br />
4. Add 2ml medium to first dish and pipette up and down to get the remaining cells stuck on the plate off<br />
5. Distribute all 4 mL between the 4 (or 5) new dishes.<br />
6. Wait three days (or until confluent) to harvest<br />
<br />
<br />
Day -1: Preparing for transfection<br />
<br />
1. Carefully suck up medium from plates<br />
2. Add 2ml of trypsin to each plate, distribute it evenly on each plate<br />
3. add more medium to each plate, like in cell splitting (medium inactivates trypsin)<br />
4. Transfer medium and cells from plate to a tube (50 ml tube). Make sure to wash all of the cells off the plate. Use as much medium as necessary but don’t make it too dilute. It should look transparent, not cloudy.<br />
<br />
Counting<br />
<br />
1. Mix tube gently by inverting up and down.<br />
2. pipette 10 ul onto a microscope slide but don’t overfill slide<br />
3. Count cells under microscope using a colony counter.<br />
4. The slide is a Reichert hemocytometer with 4x4 corners and 5x5 middle. All large squares have the same area.<br />
5. Cells/ml = # cells counted * dilution factor * 10^4<br />
<br />
Count the number of cells per large square (4x4 or 5x5). They all have the same area. For cells that overlap between squares, count only those on two sides (eg. left and top).<br />
<br />
Day -1: Plating<br />
1. Coat 3 plates/plasmid made with 0.1% gelatin in Ultra Pure water (15-20 ml). Cover for at least an hour at room temperature.<br />
2. Remove the liquid, the gelatin should stick to the plate.<br />
3. Add ~20 ml of medium to the plates<br />
4. You want to plate 1.8-2.0 * 10^7 cells per 15 cm dish<br />
5. With extra cells that are left in the tube, replate to continue growth on a clean plate, also add ~25 ml medium to the plates<br />
<br />
Day 0: Transfection<br />
1. We have three 15 cm dishes per transducing plasmid.<br />
2. Remove the medium from the plates and add carefully and slowly 12.5 ml of new growth/culture medium to each plate. Tip the plate and add the new medium gently to the side of the plate. You do not want to disturb the cells growing on the plate.<br />
3. For each 15 cm plate, use:<br />
a. 18.9 ug of p148, <br />
b. 9.5 ug of p149<br />
c. 7.43 ug of transducing vector<br />
d. and enough DMEM to make a total volume of 1110 ul<br />
<br />
Add the DMEM First!<br />
Mix well and add 75 ul of SuperFect Transfection Reagent <br />
4. Mix gently and incubate for 10 minutes at room temperature<br />
5. Add the mixture dropwise and spread evenly across the plate. After all the medium is added, give it a VERY gentle swirl.<br />
<br />
Day 1: If possible, move dish from 37 C / 5%CO2 to 32 C / 5%CO2. This step will extend the half-life of the viral particles and, hence, increase the titer.<br />
<br />
Day 2: Viral Harvesting<br />
1. Filter the supernatant from the transfected cells. Put 3 plates worth of cells in a “low protein binding membrane filter” (Corning 430625).<br />
2. Put liquid in polycarbonate centrifuge bottles, balance them perfectly by adding medium (DMEM).<br />
3. Spin at 50,000 g for 2.5 hours at 4 C, accelerate at max. Leave break off so as not to disturb the pellet of virus. It will take about 30-40 minutes for the centrifuge to stop spinning.<br />
<br />
Storage<br />
1. Carefully suck out medium without disturbing viral pellet. Tilt tube with pellet facing the ground, suck from the top. When liquid is past the level of the pellet tilt more and try to suck out all of the medium.<br />
2. Add about 80 uL PBS (Phosphate Buffered Saline), resuspend by flicking with finger. If you see virus particles floating around, pipette up and down.<br />
3. Put in 4 C fridge for 30 min<br />
4. Divide into 5 tubes or more depending on pellet size, label and date.<br />
5. Try to get everything into tubes ~20 ul per.<br />
6. Add bleach to centrifuge tubes, also bleach all tips – bleach everything in contact with virus. Let stay at least 20 minutes. Then wash the tubes out with water followed by 80% ethanol (“sterilization”), and sterile water or PBS. Dry tubes.</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-27T03:14:28Z<p>Jmonk: /* Bio-Brick Verification: Virus Harvest */</p>
<hr />
<div><html><br />
<img src="http://img341.imageshack.us/img341/8195/logo5oz0.jpg" alt="LOGO"><br />
</html> [[Princeton/overview | Overview]] || [[Princeton/People | People ]] || [[Princeton/lab | Lab work]] || [[Princeton/lab/experimentation | Experiments]] || [[Princeton/lab/bioinformatics | Bioinformatics]] || [[Princeton/lab/simulation | Simulations]]<br />
=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
=Assembly Line Flow=<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
[[PCR]]<br />
<br />
[[Run Gel]]<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction<br />
<br />
[[Digestion with Restriction Enzymes]]<br />
<br />
[[CIP Treatment]]<br />
<br />
[[PCR purification]]<br />
<br />
[[Ligation]]<br />
<br />
[[Transform and pick colonies]]<br />
<br />
[[Miniprep and determine concentrations]]<br />
<br />
[[Restriction Map]]<br />
<br />
[[Retransform with chosen plasmid]]<br />
<br />
[[Maxiprep and determine concentraions]]<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
Please see [[Making A Lentivirus]] for protocols<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Retransform_with_chosen_plasmidRetransform with chosen plasmid2007-10-27T03:12:53Z<p>Jmonk: </p>
<hr />
<div><br />
== Maxiprep transformation ==<br />
<br />
<br />
Make sure that the incubator (30/37C) and water bath (42C) are ON <br />
<br />
Make sure required antibiotic plates are present. Check the antibiotic resistance on the plasmid map in Vector NTI. <br />
<br />
Take the DNA out of -20 frig, let it thaw <br />
<br />
Thaw the competent cells on ice for 7-8 min. <br />
<br />
Add 1.0 µl of DNA (about 10ng) into the liquid (Don’t vortex). Tap the sides of the tube to mix. <br />
<br />
Incubate the cells on ice for 30 min <br />
<br />
Heat shock the cells for EXACTLY 30 sec at 42 C water bath. <br />
<br />
Place on ice for 2 min. <br />
<br />
Add 0.9ml of 37C S.O.C medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.) <br />
<br />
Shake the tubes at 37 C, 280 rpm for 60 min or 30 C for 90 min <br />
<br />
Incubate upright for 20 min., then upside down overnight (12-14 h) at 37 C or 16-18h at 30C. <br />
<br />
Can leave the cells in the incubator for up to 18 hours but no more<br />
<br />
<br />
== Maxiprep growth culture ==<br />
<br />
<br />
Calculate the approx. S/N ratio of the transformation. <br />
<br />
Decide on number of tries to be setup accordingly. Pick 12 tries if the S/N ratio is good (5:1), more otherwise. <br />
<br />
Aliquot X ml (X = Y number of tries * 3.5, where Y = number of tries.) of TB into a clean (autoclaved) beaker or flask. Lb can be used if TB is not available. <br />
<br />
Add X µl of appropriate antibiotic, i.e. a 1:1000 dilution to the above solution. The correct antibiotic should be added (same as the plate from which the colonies are picked.) <br />
<br />
Take Y clean and autoclaved 14 ml polystyrene tubes (ones with thw white cap) and place them on a rack. Label them clearly. Aliquot 3.5 ml of TB/LB into each 14 ml tube. <br />
<br />
Use a sterile wooden applicator (autoclaved) or pipette tip to carefully pick an individual colony and dip the colony end of the applicator / tip into a 14 ml tube. Repeat this Y times using a fresh applicator each time. Make sure to pick a single colony per try. <br />
<br />
Place the tubes in the 37C shaker at 280-300 rpm and grow the cells for 12-14 hours. The tubes should be murky after the overnight growth.<br />
<br />
<br />
== Maxiprep DNA extract ==<br />
<br />
<br />
Transfer 200ml of the overnight culture of plasmid cells into a clean, autoclaved 200ml centrifuge bottle (1 per plasmid). Pellet for 5 min. Decant all the liquid and add the remaining 200ml of each plasmid culture into the corresponding bottle. Make sure not to mix up the plasmids. Spin down again for 5 mins. And throw away the supernatant.<br />
<br />
Add 15ml of Resuspension solution to the cell pellet and vortex the bottle. No cell clumps should be observed. <br />
<br />
Add 23ml of Cell Lysis solution. Do not vortex. Mix gently by swirling the bottle. The solution should become slightly clear and very viscous at this stage. <br />
<br />
Add 15ml of Neutralization solution. Do not vortex. Mix gently by swirling the bottle. A white precipitate will appear. <br />
<br />
Spin down the cells for 25 mins. While this is being done, mix the bottle labeled ‘matrix solution’ well by vortexing till you have a homogenous solution. <br />
<br />
The supernatant at this stage contains the plasmid DNA. So, do not discard the supernatant. Carefully transfer the supernatant into a fresh 200 ml bottle. Do not transfer any of the precipitate. Make sure not to mix up the supernatants and label all bottles at each stage. <br />
<br />
Add 10ml of the matrix per bottle. Swirl and mix for 30 sec. Centrifuge for 5 min. The matrix binds the DNA and settles at the bottom. <br />
<br />
Throw out the supernatant. Resuspend the matrix in 25 ml of wash buffer. Centrifuge for 5 min. Make sure that the wash buffer bottle is marked ‘ethanol added’. If not, add 100% ethanol to fill the wash buffer bottle. <br />
Assemble the filters. Place each filter provided in the kit in a 50ml centrifuge tube (blue cap, also provided in the kit). Label the tubes. <br />
<br />
Throw out the supernatant. Resuspend the wash buffer in 15 ml of the wash buffer and mix thoroughly. Transfer the solution to the corresponding filter/tube from the above step. <br />
<br />
Centrifuge for 5 min. <br />
<br />
Remove the filter and throw out the solution. Place the filter back in the same tube. Add 10 ml of wash buffer and centrifuge for 5 min. <br />
<br />
Remove the filter and place it in a new 50 ml centrifuge tube (provided in the kit). Label the tube and add 4 ml of EB (elution buffer) to the filter. Let it stand for 3 min at RT.<br />
<br />
Centrifuge for 5 min. <br />
<br />
Discard the filters from each tube. Add 222 µl of 5M NaCl to each tube. Mix well by swirling gently. Add 8ml of ice cold 100% ethanol to each tube. Centrifuge at speeds > 10000g for 15 min. <br />
<br />
You will see a translucent white DNA precipitate at the bottom. Carefully throw away the supernatant without disturbing the pellet. <br />
<br />
Add 10 ml of 70% ethanol to each tube. Centrifuge at speeds > 10000g for 15 min. <br />
<br />
Once again, you will see a translucent white DNA precipitate at the bottom. Carefully throw away the supernatant without disturbing the pellet.<br />
<br />
Air dry the pellet (leave it on your bench for 10-15 min.) Don’t leave the pellet for a longer duration as it might be difficult to resuspend. <br />
<br />
Resuspend the pellet in 1.5 ml of EB. <br />
<br />
Measure the concentration and aliquot into labeled 2ml Eppendorf tubes. <br />
<br />
Store the DNA at -20C. <br />
<br />
Ultracentrifuge, 120mm<br />
<br />
6000, 6682 <br />
<br />
10000, 8626 <br />
<br />
15000, 10564 <br />
<br />
20000, 12199 <br />
<br />
Ultracentrifuge, 65mm<br />
<br />
20000, 16575 <br />
<br />
15000, 14354 <br />
<br />
Centrifuge, 185mm<br />
<br />
6000, 5381 <br />
<br />
10000, 6947 <br />
<br />
Equipment to bring to measure OD:<br />
<br />
p200, p20, tips <br />
<br />
Blanking solution, alcohol wash bottle, DI water wash bottle. <br />
<br />
Tray of samples/unknowns and required number of Eppendorfs.</div>Jmonkhttp://2007.igem.org/wiki/index.php/Retransform_with_chosen_plasmidRetransform with chosen plasmid2007-10-27T03:10:18Z<p>Jmonk: </p>
<hr />
<div>Make sure that the incubator (30/37C) and water bath (42C) are ON <br />
<br />
Make sure required antibiotic plates are present. Check the antibiotic resistance on the plasmid map in Vector NTI. <br />
<br />
Take the DNA out of -20 frig, let it thaw <br />
<br />
Thaw the competent cells on ice for 7-8 min. <br />
<br />
Add 1.0 µl of DNA (about 10ng) into the liquid (Don’t vortex). Tap the sides of the tube to mix. <br />
<br />
Incubate the cells on ice for 30 min <br />
<br />
Heat shock the cells for EXACTLY 30 sec at 42 C water bath. <br />
<br />
Place on ice for 2 min. <br />
<br />
Add 0.9ml of 37C S.O.C medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.) <br />
<br />
Shake the tubes at 37 C, 280 rpm for 60 min or 30 C for 90 min <br />
<br />
Incubate upright for 20 min., then upside down overnight (12-14 h) at 37 C or 16-18h at 30C. <br />
<br />
Can leave the cells in the incubator for up to 18 hours but no more<br />
<br />
<br />
Calculate the approx. S/N ratio of the transformation. <br />
<br />
Decide on number of tries to be setup accordingly. Pick 12 tries if the S/N ratio is good (5:1), more otherwise. <br />
<br />
Aliquot X ml (X = Y number of tries * 3.5, where Y = number of tries.) of TB into a clean (autoclaved) beaker or flask. Lb can be used if TB is not available. <br />
<br />
Add X µl of appropriate antibiotic, i.e. a 1:1000 dilution to the above solution. The correct antibiotic should be added (same as the plate from which the colonies are picked.) <br />
<br />
Take Y clean and autoclaved 14 ml polystyrene tubes (ones with thw white cap) and place them on a rack. Label them clearly. Aliquot 3.5 ml of TB/LB into each 14 ml tube. <br />
<br />
Use a sterile wooden applicator (autoclaved) or pipette tip to carefully pick an individual colony and dip the colony end of the applicator / tip into a 14 ml tube. Repeat this Y times using a fresh applicator each time. Make sure to pick a single colony per try. <br />
<br />
Place the tubes in the 37C shaker at 280-300 rpm and grow the cells for 12-14 hours. The tubes should be murky after the overnight growth.</div>Jmonkhttp://2007.igem.org/wiki/index.php/Restriction_MapRestriction Map2007-10-27T03:08:48Z<p>Jmonk: </p>
<hr />
<div>Choose a restriction enzyme such that the enzyme(s) cut both the vector and the insert and the bands can be distinguished reasonably on a gel to identify the correct try. Vector NTI should be used to design restriction maps. <br />
<br />
Make sure to create a gel map on vector for the restriction digest. Also remember to digest the parents with the same enzyme(s). <br />
<br />
Restriction maps should be setup just like digestions are setup. Carefully calculate the amount of DNA you would need to digest. You need at least 50ng of DNA per band to see it on the gel. The DNA for each band is proportional to its size : for example if you expect to see a 500 bp band and a 4.5 kb out of a 5 kb plasmid after your digest, you need to digest atleast 0.5 µg as they will be divided as 450ng of the 4.5kb band and 50ng of the 500 bp band. <br />
<br />
Keep the total volume as low as possible. <br />
<br />
Make gels as before ([[gel extraction]] protocol). <br />
<br />
Run on the agarose gel for as long as required to obtain maximum resolution. <br />
<br />
Compare with the expected pattern of bands and pick out the correct try(ies) for maxiprep transformation</div>Jmonkhttp://2007.igem.org/wiki/index.php/Restriction_MapRestriction Map2007-10-27T03:08:46Z<p>Jmonk: </p>
<hr />
<div>Choose a restriction enzyme such that the enzyme(s) cut both the vector and the insert and the bands can be distinguished reasonably on a gel to identify the correct try. Vector NTI should be used to design restriction maps. <br />
<br />
Make sure to create a gel map on vector for the restriction digest. Also remember to digest the parents with the same enzyme(s). <br />
<br />
Restriction maps should be setup just like digestions are setup. Carefully calculate the amount of DNA you would need to digest. You need at least 50ng of DNA per band to see it on the gel. The DNA for each band is proportional to its size : for example if you expect to see a 500 bp band and a 4.5 kb out of a 5 kb plasmid after your digest, you need to digest atleast 0.5 µg as they will be divided as 450ng of the 4.5kb band and 50ng of the 500 bp band. <br />
<br />
Keep the total volume as low as possible. <br />
<br />
Make gels as before (gel extraction protocol). <br />
<br />
Run on the agarose gel for as long as required to obtain maximum resolution. <br />
<br />
Compare with the expected pattern of bands and pick out the correct try(ies) for maxiprep transformation</div>Jmonkhttp://2007.igem.org/wiki/index.php/Miniprep_and_determine_concentrationsMiniprep and determine concentrations2007-10-27T03:07:28Z<p>Jmonk: /* Miniprep Protocol */</p>
<hr />
<div><br />
== Miniprep Protocol ==<br />
<br />
<br />
Calculate the approx. S/N ratio of the transformation. <br />
<br />
Decide on number of tries to be setup accordingly. Pick 12 tries if the S/N ratio is good (5:1), more otherwise. <br />
<br />
Aliquot X ml (X = Y number of tries * 3.5, where Y = number of tries.) of TB into a clean (autoclaved) beaker or flask. Lb can be used if TB is not available. <br />
<br />
Add X µl of appropriate antibiotic, i.e. a 1:1000 dilution to the above solution. The correct antibiotic should be added (same as the plate from which the colonies are picked.) <br />
<br />
Take Y clean and autoclaved 14 ml polystyrene tubes (ones with thw white cap) and place them on a rack. Label them clearly. Aliquot 3.5 ml of TB/LB into each 14 ml tube. <br />
<br />
Use a sterile wooden applicator (autoclaved) or pipette tip to carefully pick an individual colony and dip the colony end of the applicator / tip into a 14 ml tube. Repeat this Y times using a fresh applicator each time. Make sure to pick a single colony per try. <br />
<br />
Place the tubes in the 37C shaker at 280-300 rpm and grow the cells for 12-14 hours. The tubes should be murky after the overnight growth.<br />
<br />
<br />
<br />
== Miniprep DNA Extraction ==<br />
<br />
<br />
Transfer the overnight culture of plasmid cells into 2ml microcentrifuge collection tubes (1 per try)provided in the kit. Pellet for 1 min. Decant all the liquid and add 1 ml of the culture into the corresponding tube. <br />
<br />
Make sure not to mix up the tries. <br />
<br />
Add 400ul of ice cold lysis buffer (make sure enzyme mix is already added. Lysis buffer is stored at 4C) <br />
Vortex for exactly 30 sec. Let it stay at RT for 3 mins.<br />
<br />
Setup filters and collection tubes. <br />
<br />
Transfer the contents to the filter + collection tube setup, each try in a separate filter. <br />
<br />
Centrifuge for 1 min. <br />
<br />
Add 400 µl of Eppendorf Fast plasmid wash buffer. (make sure isopropanol is already added). Spin for 1 min. You need not dicard the contents before this step. <br />
<br />
Remove the Spin filter from the tube, discard the filtrate and put the tube back. Spin for 1 min to get rid of the residual alcohol.<br />
<br />
Transfer the filters to a new clean and autoclaved 2ml eppendorf tube. <br />
<br />
Label the tubes. <br />
Add 50 µl of EB per column and wait 3 min. <br />
<br />
Spin for 5 mins. <br />
<br />
Measure the concentration of DNA. <br />
<br />
Digest the tries which seem to have the correct supercoil size with an appropriate restriction enzyme to verify plasmid construction. Make sure to create a gel map on vector for the restriction digest. Also remember to digest the parents with the same enzyme. You need atleast 100ng of DNA per band to see it on the gel. The DNA for each band is proportional to its size : for example if you expect to see a 500 bp band and a 4.5 kb out of a 5 kb plasmid after your digest, you need to digest atleast 1 µg as they will be divided as 900ng of the 4.5kb band and 100ng of the 500 bp band. <br />
<br />
Run on the agarose gel for as long as required to obtain maximum resolution.</div>Jmonkhttp://2007.igem.org/wiki/index.php/Miniprep_and_determine_concentrationsMiniprep and determine concentrations2007-10-27T03:06:35Z<p>Jmonk: </p>
<hr />
<div><br />
== Miniprep Protocol ==<br />
<br />
<br />
Calculate the approx. S/N ratio of the transformation. <br />
<br />
Decide on number of tries to be setup accordingly. Pick 12 tries if the S/N ratio is good (5:1), more otherwise. <br />
<br />
Aliquot X ml (X = Y number of tries * 3.5, where Y = number of tries.) of TB into a clean (autoclaved) beaker or flask. Lb can be used if TB is not available. <br />
<br />
Add X µl of appropriate antibiotic, i.e. a 1:1000 dilution to the above solution. The correct antibiotic should be added (same as the plate from which the colonies are picked.) <br />
<br />
Take Y clean and autoclaved 14 ml polystyrene tubes (ones with thw white cap) and place them on a rack. Label them clearly. Aliquot 3.5 ml of TB/LB into each 14 ml tube. <br />
<br />
Use a sterile wooden applicator (autoclaved) or pipette tip to carefully pick an individual colony and dip the colony end of the applicator / tip into a 14 ml tube. Repeat this Y times using a fresh applicator each time. Make sure to pick a single colony per try. <br />
<br />
Place the tubes in the 37C shaker at 280-300 rpm and grow the cells for 12-14 hours. The tubes should be murky after the overnight growth.</div>Jmonkhttp://2007.igem.org/wiki/index.php/Transform_and_pick_coloniesTransform and pick colonies2007-10-27T03:05:53Z<p>Jmonk: </p>
<hr />
<div>== Heat Transformation ==<br />
<br />
Make sure that the incubator (30/37C) and water bath (42C) are ON <br />
<br />
Make sure required antibiotic plates are present. Check the antibiotic resistance on the plasmid map in Vector NTI. <br />
<br />
Take the DNA out of -20 frig, let it thaw <br />
<br />
Thaw the competent cells on ice for 7-8 min. <br />
<br />
Add 1.0 µl of DNA (about 10ng) into the liquid (Don’t vortex). Tap the sides of the tube to mix. <br />
<br />
Incubate the cells on ice for 30 min <br />
<br />
Heat shock the cells for EXACTLY 30 sec at 42 C water bath.<br />
<br />
Place on ice for 2 min. <br />
<br />
Add 0.9ml of 37C S.O.C medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.) <br />
<br />
Shake the tubes at 37 C, 280 rpm for 60 min or 30 C for 90 min <br />
<br />
Incubate upright for 20 min., then upside down overnight (12-14 h) at 37 C or 16-18h at 30C. <br />
<br />
Can leave the cells in the incubator for up to 18 hours but no more</div>Jmonkhttp://2007.igem.org/wiki/index.php/PCR_purificationPCR purification2007-10-27T03:04:50Z<p>Jmonk: </p>
<hr />
<div>Using QIAquick kit<br />
<br />
Add 5 volumes of Buffer PBI to 1 volume of sample. <br />
<br />
Pipette into a QIAquick spin column(max 770 µl) and centrifuge for 60 sec at 10,000g <br />
<br />
Discard flow-through. <br />
<br />
Wash: add 0.75ml Buffer PE(make sure that the buffer has ethanol added to it) to column and centrifuge for 1 min <br />
<br />
Discard flow-through & centrifuge for 1 min <br />
<br />
Place column into clean Eppendorf tube <br />
<br />
Add 50ul Buffer EB or water to center of membrane <br />
<br />
Let stand at RT for 3 min <br />
<br />
Centrifuge for 5 min. <br />
<br />
Measure the concentration using the UV spectrophotometer.</div>Jmonkhttp://2007.igem.org/wiki/index.php/CIP_TreatmentCIP Treatment2007-10-27T03:04:11Z<p>Jmonk: </p>
<hr />
<div>CIP treatment is done to phosphatase the vector used for plasmid ligations. This is done to reduce the self ligation of a vector digested with enzyme(s) creating compatible sticky ends and hence enhance the S/N ratio of transformations.<br />
<br />
Use 10 units of CIP per 1µg of DNA (over digesting by factor of X) <br />
<br />
Calculate volumes <br />
<br />
DNA µg = DNA volume * concentration <br />
<br />
Enzyme volume = Enzyme unit/µl* # units = X [µl] <br />
<br />
Buffer is dilution factor x dilution of the total volume. <br />
<br />
[i.e. for 10X over digest - buffer is 10%, 3x - 30% of total volume] <br />
Order of filling <br />
<br />
DNA <br />
<br />
Water <br />
<br />
Buffer <br />
<br />
CIP <br />
<br />
Incubate for 1 hours at the specified temperature for the enzyme (37C). <br />
Keep the buffer on ice and the CIP in the benchtop coolers when on the bench.</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-27T03:03:38Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
<div><html><br />
<img src="http://img341.imageshack.us/img341/8195/logo5oz0.jpg" alt="LOGO"><br />
</html> [[Princeton/overview | Overview]] || [[Princeton/People | People ]] || [[Princeton/lab | Lab work]] || [[Princeton/lab/experimentation | Experiments]] || [[Princeton/lab/bioinformatics | Bioinformatics]] || [[Princeton/lab/simulation | Simulations]]<br />
=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
=Assembly Line Flow=<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
[[PCR]]<br />
<br />
[[Run Gel]]<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction<br />
<br />
[[Digestion with Restriction Enzymes]]<br />
<br />
[[CIP Treatment]]<br />
<br />
[[PCR purification]]<br />
<br />
[[Ligation]]<br />
<br />
[[Transform and pick colonies]]<br />
<br />
[[Miniprep and determine concentrations]]<br />
<br />
[[Restriction Map]]<br />
<br />
[[Retransform with chosen plasmid]]<br />
<br />
[[Maxiprep and determine concentraions]]<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-27T03:03:02Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
<div><html><br />
<img src="http://img341.imageshack.us/img341/8195/logo5oz0.jpg" alt="LOGO"><br />
</html> [[Princeton/overview | Overview]] || [[Princeton/People | People ]] || [[Princeton/lab | Lab work]] || [[Princeton/lab/experimentation | Experiments]] || [[Princeton/lab/bioinformatics | Bioinformatics]] || [[Princeton/lab/simulation | Simulations]]<br />
=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
=Assembly Line Flow=<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
[[PCR]]<br />
<br />
[[Run Gel]]<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction<br />
<br />
[[Digestion with Restriction Enzymes]]<br />
<br />
[[CIP]]<br />
<br />
[[PCR purification]]<br />
<br />
[[Ligation]]<br />
<br />
[[Transform and pick colonies]]<br />
<br />
[[Miniprep and determine concentrations]]<br />
<br />
[[Restriction Map]]<br />
<br />
[[Retransform with chosen plasmid]]<br />
<br />
[[Maxiprep and determine concentraions]]<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Run_GelRun Gel2007-10-27T03:01:43Z<p>Jmonk: </p>
<hr />
<div><br />
== Preparing the Gel ==<br />
<br />
Dissolve UltraPure agarose to a final concentration of 1% in TAE buffer in a glass bottle. Heat the solution in the microwave with frequent stirring to dissolve the agarose homogenously. Place the solution in a 55C water bath for 15 mins. Add 1 µl EtBr (Ethidium Br;komide) per 50 ml of the solution and mix well. Pour 50ml of solution per small gel tray. (the gel trays and combs should be pre-cleaned with water and wiped dry). Wait for the gels to solidify. Label and store at 4C.<br />
<br />
Use 120ml per large gel tray. Insert one large and one small comb in each small gel tray.<br />
<br />
<br />
== Running the Gel ==<br />
<br />
There are two kinds of wells that fit different volumes:<br />
<br />
Small: 15 ul <br />
<br />
Big: 40 ul <br />
<br />
Appropriate Hyperladder to be used for PCR product which is linear. Usually Hyperladder I will be used. 1. Add gel loading buffer (Orange G 6X), add 1X to 5X of DNA (it helps DNA sink into the bottom of the well) to DNA. 2. Make sure there is enough 1xTAE in the plate holder. 3. Load 5ul of appropriate hyperladder to one of the lanes. 4. Load appropriate amount of DNA (mixed with the buffer) in each well. 5. Set the timer and voltage to 120V and 60 min.<br />
<br />
== Gel Extraction Protocol using QIAquick Gel Extraction Kit: ==<br />
<br />
Look at the gel under low wavelength UV (high wavelengths will denature DNA). Quickly take a polaroid image, cut out relevant bands and shut OFF the UV. <br />
<br />
Place the cut bands in 2ml Eppendorf tubes; Weigh slices; No more than 400mg per tube <br />
Add 3 volumes of Buffer QG to 1 volume of gel (100mg ~ 100ul) <br />
<br />
Incubate at 50C for 10min or until gel is dissolved; vortex every 2-3 min <br />
<br />
Confirm that color of mixture is yellow (if not, add 10ul of 3M NaAc, pH 5.0) <br />
<br />
Add 1 gel volume of isopropanol (not necessary for DNA fragments between 500-4000bp) <br />
<br />
Add max of 770ul to QIAquick column and centrifuge for 1 min (max speed, ~13,000rpm, RT) <br />
<br />
Discard flow-through and place column back in tube. <br />
<br />
If needed, add rest of mixture to same tube (up to additional 770ul), spin, and discard flow-through <br />
<br />
Wash: add 0.75ml Buffer PE(make sure that the buffer has ethanol added to it) to column and centrifuge for 1 min <br />
<br />
Discard flow-through & centrifuge for 1 min <br />
<br />
Place column into clean Eppendorf tube <br />
<br />
Add 50ul Buffer EB or water to center of membrane <br />
<br />
Let stand at RT for 3 min <br />
<br />
Centrifuge for 5 min and throw away the column. <br />
<br />
Measure the concentration using the UV spectrophotometer.</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-27T03:00:46Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
<div><html><br />
<img src="http://img341.imageshack.us/img341/8195/logo5oz0.jpg" alt="LOGO"><br />
</html> [[Princeton/overview | Overview]] || [[Princeton/People | People ]] || [[Princeton/lab | Lab work]] || [[Princeton/lab/experimentation | Experiments]] || [[Princeton/lab/bioinformatics | Bioinformatics]] || [[Princeton/lab/simulation | Simulations]]<br />
=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
=Assembly Line Flow=<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
[[PCR]]<br />
<br />
[[Run Gel]]<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction<br />
<br />
[[Digestion with Restriction Enzymes]]<br />
<br />
[[PCR purification]]<br />
<br />
[[Ligation]]<br />
<br />
[[Transform and pick colonies]]<br />
<br />
[[Miniprep and determine concentrations]]<br />
<br />
[[Restriction Map]]<br />
<br />
[[Retransform with chosen plasmid]]<br />
<br />
[[Maxiprep and determine concentraions]]<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-27T02:59:59Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
<div><html><br />
<img src="http://img341.imageshack.us/img341/8195/logo5oz0.jpg" alt="LOGO"><br />
</html> [[Princeton/overview | Overview]] || [[Princeton/People | People ]] || [[Princeton/lab | Lab work]] || [[Princeton/lab/experimentation | Experiments]] || [[Princeton/lab/bioinformatics | Bioinformatics]] || [[Princeton/lab/simulation | Simulations]]<br />
=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
=Assembly Line Flow=<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
[[PCR]]<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction<br />
<br />
[[Digestion with Restriction Enzymes]]<br />
<br />
[[PCR purification]]<br />
<br />
[[Ligation]]<br />
<br />
[[Transform and pick colonies]]<br />
<br />
[[Miniprep and determine concentrations]]<br />
<br />
[[Restriction Map]]<br />
<br />
[[Retransform with chosen plasmid]]<br />
<br />
[[Maxiprep and determine concentraions]]<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Digestion_with_Restriction_EnzymesDigestion with Restriction Enzymes2007-10-27T02:59:08Z<p>Jmonk: </p>
<hr />
<div>== Vector digestion ==<br />
<br />
<br />
Use X units of enzymes per 1ug of DNA (over digesting by factor of X) Usually we use over digest factor of 10 unless otherwise specified in the enzyme tech sheet. If over digestion results in star activity use 3X <br />
<br />
Calculate the volume required for each <br />
<br />
DNA µg = DNA volume * concentration <br />
<br />
Enzyme volume = Enzyme unit/µl* # units = X [µl] <br />
Buffer is dilution factor x dilution of the total volume. [i.e. for 10X over digest - buffer is 10%, 3x - 30% of total volume] <br />
Order of filling <br />
<br />
DNA <br />
<br />
Water <br />
<br />
Buffer <br />
<br />
Enzyme <br />
<br />
Incubate for 3 hours at the specified temperature for the enzyme and deactivate at the appropriate temperature for 20 mins. <br />
<br />
Please keep the buffer on ice at all times when out of -20C. Keep the enzyme in the benchtop coolers at all times when out of the -20C.</div>Jmonkhttp://2007.igem.org/wiki/index.php/Digestion_with_Restriction_EnzymesDigestion with Restriction Enzymes2007-10-27T02:57:52Z<p>Jmonk: </p>
<hr />
<div><br />
== Vector digestion ==<br />
<br />
<br />
Use X units of enzymes per 1ug of DNA (over digesting by factor of X) Usually we use over digest factor of 10 unless otherwise specified in the enzyme tech sheet. If over digestion results in star activity use 3X <br />
Calculate the volume required for each <br />
DNA µg = DNA volume * concentration <br />
Enzyme volume = Enzyme unit/µl* # units = X [µl] <br />
Buffer is dilution factor x dilution of the total volume. [i.e. for 10X over digest - buffer is 10%, 3x - 30% of total volume] <br />
Order of filling <br />
DNA <br />
Water <br />
Buffer <br />
Enzyme <br />
Incubate for 3 hours at the specified temperature for the enzyme and deactivate at the appropriate temperature for 20 mins. <br />
Please keep the buffer on ice at all times when out of -20C. Keep the enzyme in the benchtop coolers at all times when out of the -20C.</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-27T02:57:30Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
<div><html><br />
<img src="http://img341.imageshack.us/img341/8195/logo5oz0.jpg" alt="LOGO"><br />
</html> [[Princeton/overview | Overview]] || [[Princeton/People | People ]] || [[Princeton/lab | Lab work]] || [[Princeton/lab/experimentation | Experiments]] || [[Princeton/lab/bioinformatics | Bioinformatics]] || [[Princeton/lab/simulation | Simulations]]<br />
=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
=Assembly Line Flow=<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
[[PCR]]<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction<br />
<br />
[[Digestion with Restriction Enzymes]]<br />
<br />
PCR purification<br />
<br />
Ligation<br />
<br />
Transform and pick colonies<br />
<br />
Miniprep and determine concentrations<br />
<br />
Restriction Map<br />
<br />
Retransform with chosen plasmid<br />
<br />
Maxiprep and determine concentraions<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-27T02:56:44Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
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<img src="http://img341.imageshack.us/img341/8195/logo5oz0.jpg" alt="LOGO"><br />
</html> [[Princeton/overview | Overview]] || [[Princeton/People | People ]] || [[Princeton/lab | Lab work]] || [[Princeton/lab/experimentation | Experiments]] || [[Princeton/lab/bioinformatics | Bioinformatics]] || [[Princeton/lab/simulation | Simulations]]<br />
=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
=Assembly Line Flow=<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
[[PCR]]<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification<br />
<br />
Ligation<br />
<br />
Transform and pick colonies<br />
<br />
Miniprep and determine concentrations<br />
<br />
Restriction Map<br />
<br />
Retransform with chosen plasmid<br />
<br />
Maxiprep and determine concentraions<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T22:21:56Z<p>Jmonk: /* Experimentation */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
=Assembly Line Flow=<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification<br />
<br />
Ligation<br />
<br />
Transform and pick colonies<br />
<br />
Miniprep and determine concentrations<br />
<br />
Restriction Map<br />
<br />
Retransform with chosen plasmid<br />
<br />
Maxiprep and determine concentraions<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T22:14:15Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
==Assembly Line Flow==<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification<br />
<br />
Ligation<br />
<br />
Transform and pick colonies<br />
<br />
Miniprep and determine concentrations<br />
<br />
Restriction Map<br />
<br />
Retransform with chosen plasmid<br />
<br />
Maxiprep and determine concentraions<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T22:13:47Z<p>Jmonk: /* Experimentation */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
==Assembly Line Flow==<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction and determine concentrations<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification and determine concentrations<br />
<br />
Ligation<br />
<br />
Transform and pick colonies<br />
<br />
Miniprep and determine concentrations<br />
<br />
Restriction Map<br />
<br />
Retransform with chosen plasmid<br />
<br />
Maxiprep and determine concentraions<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T22:13:31Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
==Assembly Line Flow==<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
<br />
- Check Primer design<br />
<br />
Gel Extraction and determine concentrations<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification and determine concentrations<br />
<br />
Ligation<br />
<br />
Transform and pick colonies<br />
<br />
Miniprep and determine concentrations<br />
<br />
Restriction Map<br />
<br />
Retransform with chosen plasmid<br />
<br />
Maxiprep and determine concentraions<br />
<br />
Sequence<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T22:13:17Z<p>Jmonk: /* Bio-Brick Verification: Quality Control */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
==Assembly Line Flow==<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
- Check Primer design<br />
<br />
Gel Extraction and determine concentrations<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification and determine concentrations<br />
<br />
Ligation<br />
<br />
Transform and pick colonies<br />
<br />
Miniprep and determine concentrations<br />
<br />
Restriction Map<br />
<br />
Retransform with chosen plasmid<br />
<br />
Maxiprep and determine concentraions<br />
<br />
Sequence<br />
<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T22:13:11Z<p>Jmonk: /* Bio-Brick Verification */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
==Assembly Line Flow==<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
- Check Primer design<br />
<br />
Gel Extraction and determine concentrations<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification and determine concentrations<br />
<br />
Ligation<br />
<br />
Transform and pick colonies<br />
<br />
Miniprep and determine concentrations<br />
<br />
Restriction Map<br />
<br />
Retransform with chosen plasmid<br />
<br />
Maxiprep and determine concentraions<br />
<br />
Sequence<br />
<br />
<br />
== Bio-Brick Verification: Virus Harvest ==<br />
<br />
Prepare cell culture media<br />
<br />
Seed cells<br />
<br />
Check health of cells<br />
<br />
Transfect<br />
<br />
Check phenotype 24hrs post transfection<br />
<br />
Transform correct try using XL-10 cells<br />
<br />
Maxiprep and ethanal purify the try and packaging plasmids<br />
<br />
Seed 293FT cells<br />
<br />
Check health of cells 24hrs later<br />
<br />
Transform the vector and packaging plasmids using the CaPO4 method<br />
<br />
Check for fluorescence after 24hrs<br />
<br />
Harvest virus and concentrate using ultra-centrifugation<br />
<br />
<br />
== Bio-Brick Verification: Quality Control ==<br />
<br />
Grow cells to confluence<br />
<br />
Seed cells<br />
<br />
Infect cells with virus<br />
<br />
Change media next day<br />
<br />
Cellular experiments<br />
<br />
- Microscopy<br />
- FACS</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T22:05:24Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
==Assembly Line Flow==<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
- Check Primer design<br />
<br />
Gel Extraction and determine concentrations<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification and determine concentrations<br />
<br />
Ligation<br />
<br />
Transform and pick colonies<br />
<br />
Miniprep and determine concentrations<br />
<br />
Restriction Map<br />
<br />
Retransform with chosen plasmid<br />
<br />
Maxiprep and determine concentraions<br />
<br />
Sequence<br />
<br />
<br />
== Bio-Brick Verification ==</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T22:05:04Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
==Assembly Line Flow==<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
- Check Primer design<br />
<br />
Gel Extraction and determine concentrations<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification and determine concentrations<br />
<br />
Ligation<br />
<br />
Transform and pick colonies<br />
<br />
Miniprep and determine concentrations<br />
<br />
Restriction Map<br />
<br />
Retransform with chosen plasmid<br />
<br />
Maxiprep and determine concentraions<br />
<br />
Sequence</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T21:58:10Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
==Assembly Line Flow==<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
- Check Primer concentration<br />
- Check Primer design<br />
<br />
Gel Extraction and determine concentrations<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification and determine concentrations<br />
<br />
Ligation</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T21:55:24Z<p>Jmonk: /* Bio-Brick Creation */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
==Assembly Line Flow==<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
Gel Extraction and determine concentrations<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification and determine concentrations<br />
<br />
Ligation</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T21:54:40Z<p>Jmonk: /* Assembly Line Flow */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
==Assembly Line Flow==<br />
<br />
<br />
== Bio-Brick Creation ==<br />
<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
Gel Extraction and determine concentrations<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification and determine concentrations<br />
<br />
Ligation</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T21:54:01Z<p>Jmonk: /* Protocols */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
==Assembly Line Flow==<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
Gel Extraction and determine concentrations<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification and determine concentrations<br />
<br />
Ligation</div>Jmonkhttp://2007.igem.org/wiki/index.php/Princeton/lab/experimentationPrinceton/lab/experimentation2007-10-26T21:53:49Z<p>Jmonk: /* Experimentation */</p>
<hr />
<div>=Experimentation=<br />
<br />
==Protocols==<br />
For reliability, a majority of our actual experimentation has followed protocols provided by the supplier of relevant projects, including enzymes ([http://www.neb.com/ NEB]) and kits ([http://www.qiagen.com/ Qiagen]). The [http://www.qiagen.com/ Qiagen] maxiprep kit was replaced with a [http://www.bio-rad.com/ Bio-Rad] kit to obtain higher yields with lower processing times.<br />
<br />
Please consult the relevant vendors for protocol documents.<br />
<br />
Assembly Line Flow<br />
<br />
Design Primers<br />
<br />
PCR<br />
<br />
Run Gel<br />
<br />
Gel Extraction and determine concentrations<br />
<br />
Digestion with Restriction Enzymes<br />
<br />
PCR purification and determine concentrations<br />
<br />
Ligation</div>Jmonk