Toronto/Lab Notebook/August

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August 31, 2007

F/T: Anam

Start time: 8 am

  • Quantitated J23100 C, S01414 AB, & S01640 AB.
  • Ligated J23100 AB to S01414 AB (respectively), J23100 C to S01640 B, and J23100 D to S01640 A.

Start time: 5:30 pm

  • Transformed J23100 A + S01414 A, J23100 B + S01414 B, J23100 C + S01640 B in DH5A cells.
    • J23100 D + S01640 A was not transformed because there are only three AMP plates.
  • MP o/n F1610 AB from the plate Seema gave us (part was from 2006 plate).
    • Incubating o/n in shaker.

TO DO:

  • MP F1610 and do PD length check for it.
  • MP o/n the transformed ligated J23100 + insert (S01414 or S01640)
    • How many colonies should we test from each plate?
  • Make AMP plates
  • Transform the other ligated J23100 D + SO1640 A

P/T: Rafsan

Start time: 6:30 pm

  • Created stocks of parts to store in -80°C freezer.

PROCEDURE:

  • Did MP o/n yesterday
  • Added 500 μL of MP o/n solution in eppendorf tube
  • Added 500 μL of Glycerol in eppendorf tube & mix well
  • Filled 5 eppendorf tubes per part

August 30, 2007

Morning Session

Start time: 2:30 pm

F/T: Yusuf

  • Since the part F1610 was not working from the iGem 2007 plate, we had to borrow this part from Seema whose F1610 part was from iGem 2006 plate and presumably it worked for her. So I did transformation for the part F1610 and left it for overnight in the incubator.
  • Tomorrow, 31st August, 2007, I have to do Mini prep o/n for F1610
  • I have filled up all the Pipette Tips and will drop off at the Autoclave room to be sterilised and we later need to pick them up tomorrow morning.
  • Things To Do for the EVENING SESSION:
    • Make stock cells for the parts that had successful length checks
    • Ligation for J23100 with S01640 and S01414
  • Things To Do TOMORROW BEFORE GROUP MEETING AT 12PM AT MED-SCI CAFETERIA:
    • DEPENDS ON HOW MUCH WE ARE DONE TODAY

Evening Session

F/T: Anam P/T: Rafsan, Fareeha

  • MP o/n for all parts except for F1610, J23100 (one colony each).
  • Performed quantitation for parts J23100 ABCD, S01414 AB, S01640 AB.
    • Got data for J23100 ABD, didnt get data for the rest because the dna was too dilute (since I accidently put in 50 μL of TE buffer) and so didnt show up on the gel (or shows extremely light.
      • TO YUSUF: I will finish up quantitation and ligation before morning team comes in tomorrow.

August 29, 2007

Morning Session

Start time: 11 am

F/T: Anam

P/T: Talal (1:30pm to 4:30pm)

  • MP J37033 colonies A’’, B’’, C’’, D’’ & F1610 colonies A’’, B’’, C’’, D’’
  • Froze pellets of J37033 colonies A’’’, B’’’, C’’’, D’’’ since doing MP on this right now would unnecessarily waste MP kit materials (Seema has expressed concern on the point that we are going through the Fermentas MP kits too fast). Will wait to fully MP this after PD length check results. If the J37033 colonies A’’, B’’, C’’, D’’ pass the length check, we don't need to MP this.
  • PD digest of J23100 ABCD (used enzymes S/P for plasmid), S01414 AB (used enzymes X/P for insert), S01640 AB (used enzymes X/P for insert). Used following recipe for PD:
    • 21 µL of plasmid, 3 µL of BSA, 3 µL of Buffer, 1.5 µL of enzyme1, 1.5 µL of enzyme2
    • Used buffer 2 for all PD.
  • Gel run for all performed.
  • Turned out okay, but the total volume for the recipe used today was a bit too much for the small wells. Next time only use the amounts given on the PD recipe on protocol multiplied by 2.
  • Extracted parts from gel, purified it, and stored it in the -20°C freezer for ligation tomorrow.
    • Note: Used 50 μL of TE buffer during gel purification instead of 30 uL. Will use 30 uL next time because it concentrates the DNA.

Evening Session

P/T: Maria and Daniel (5 pm)

  • PD of J37033 colonies A’’, B’’, C’’, D’’ & F1610 colonies A’’, B’’, C’’, D’’ using enzymes X/S
  • Ran gel for PD length check

Results

F1610 (for all colonies): Band 1 -> 6-7 OR 7, Band 2 -> 14

  • Saw plasmid, but not part even though we should have been able to see it. Saw a band of a very small size which was unusual and unexpected.

J37033 (for all colonies): 8-9 OR 9

  • Saw only plasmid, but not part even though we should have been able to see it.

TO DO (regarding PD length check)

  • Check this unexpected part of small size is actually a subpart of F1610.
  • Start looking for subparts to build F1610 from.
  • J37033 has two other working analogs which can substitute for it (S01414, S01640)

August 28, 2007

Morning Session

Start time: 11:00 a.m. - End time: 2:30 p.m.

Members present: Yusuf, Talal

  1. Carried out mini-prep of F1610
    • Four colonies labeled A’ , B’ , C’ and D’ were selected
    • Then, plasmid digestion and subsequent length check was carried out
    • Results: 1st band (bright) between 5 & 6 (right in the middle). This band makes sense for part + plasmid. 2nd band (faint) between 13 & 14 (closer to 13). Doesn’t make sense.
  2. Miniprep overnight carried out for J37033
    • Four colonies A’’, B’’, C’’, and D’’ were selected and incubated overnight in shaker.

Evening Session

Start time: 5pm

Members: Anam, Daniel, Maria

  • MP o/n for J37033 using newly transformed cells (this part is giving us trouble…maybe if we check a lot of different colonies at the same time, we might get lucky since it increases our chance of finding the sample from the right colony)
  • Four colonies A’’’, B’’’, C’’’, D’’’ incubated overnight in shaker
  • MP o/n of F1610 using different colonies from the same plate that had been transformed before by Yusuf and Charles
  • Four colonies labeled A’’, B’’, C’’, D’’ incubated in shaker overnight.
  • Made 1 X TAE Buffer since we were out.

August 27, 2007

Morning Session

Start Time: 11:00 pm – End Time: 3:00 pm

Members Present: Yusuf, Mimi, Talal

  • Carried out plasmid digest (length checks) on I 13507 B and J 37033 B
  • Length Check results:
    • I 13507 B – Overall 3 bands were seen for this plasmid
      • 6-7 (Closer to 7 than to 6)
      • 8-9 (Closer to 9 than to 8)
      • 11-12 (Closer to 11 than to 12)
      • The number of parts and parts and plasmid match up closely but the first band between 6-7 could be of the uncut plasmid
    • J 37033 B – Overall 2 bands were seen
      • 8-9 (Closer to 9 than to 8)
      • 10-11 (In the middle)
      • The bands formed doesn’t match up with the given numbers for its part and plasmid from the registry
  • Did Mini-prep overnight for F 1610 with 4 colonies labeled A', B', C', D' and put it in the incubator

Evening Session

Start time: 5pm - End time: 9:30 pm

F/T: Anam

  • Performed DH5(alpha) + J37033 transformation since none of the PD length checks for all four colonies was odd and the last transformation showed only 5 big, dense circles with very tiny dots around each one even though it was not left for more time than the other transformation. This odd growth might be due to a problem with the transformation. Possible fungus growth? (Seema has informed me that there is a chance that they could grow during the transformation)


Plasmid Length Check Results
  I13507 F1610 S01640 S01414 J37033¹ J23100² S01003
A 1000-750 (saw 3 bands) 4000-3500, 500-250 1000-750 1000-750 5000-4000, 2000-1500 3000 1000-750 (saw 3 bands)
B 3500-2500, 2500-2000, 1000-750 3500-2500, 500-250 1000-750 1000-750 (saw 3 bands) 2000-1500, 1500-1000 3000-2500 3000-2500 (faint), 2000 (brightest), 1500-1000 (2nd brightest)
C 3000-2500³, 2500-2000, 1000-750 (middle) 3000-2500(middle), 2000-1500 OR 2500-2000, 500-250 3000-2500, 2500-2000, 1000-750 2500-2000, 1000-750 2500-2000 3500-3000 3000-2500 (faint), 2000 (brightest), 1500-1000 (2nd brightest)
D 2500-2000, 1000-750 (middle) 3000-2500 (middle), 500-250 3000-2500, 2500-2000, 1000-750 2500-2000, 1000-750 2500-2000 3000-2500 3000-2500 (faint), 2000 (brightest), 1500-1000 (2nd brightest)

Legend:

  • / - means "in between"
  • Boldfaced – means "closer to"
  • ¹ For Part J37033 colonies C and D, only one band was seen.
  • ² Cannot see part, but saw plasmid for C and D for which Yusuf cut the part out
  • ³ One person this while the other didn’t


Note from Anam: For colonies A of all parts, S02640 colony B, S01414 colony B, and F1610 colony B, I only wrote down the location of the band that had run furthest in the gel (except for the ones that weren’t correct – I recorded all the band locations for troubleshooting later) because all the parts are significantly smaller than their corresponding plasmids (Mistake on my part..I will write all the band locations in the future). I just wrote down how many bands I saw if there were more than 2 there.


CONCLUSION from length check

All parts passed except for all colonies of F1610 and J37033.

August 24, 2007

Evening Session

Time: 5 pm

F/T: Anam

P/T: Talal

  • Record the results of the gel Yusuf had made and run (included in table below)
  • PD length check for J23100 colony C successful.
  • 1 band between 6 and 7 on gel

August 23, 2007

Evening Session

Start time: 6:30pm

F/T: Anam

P/T: Fareeha

  • Done length check PD for all of A, green circle B, red circle B, black dot B
  • Enzymes X and P used for all except for J23100 because the part is too small and so the plasmid that contained it was linearized using enzyme P.
  • Buffer 2 was used.

Results

  • All were correct length except for samples from colonies A and B of F1610 and colony A of J37033.
  • S01003 (colony A), S01414 (colony B), I13507 (colony A) had 3 bands.
  • The 3rd band might have shown up because some of the plasmid was only cut in one location.

To do list

  1. Do a PD length check for the rest of the samples
  2. Discuss with Yusuf how much information needs to be recorded when observing the bands in a gel for PD length check

Note: There is one gel left in the fridge. It was made today.

August 22, 2007

Evening Session

Start Time: 5:30 pm – End Time: 7:30 pm

Members Present: Yusuf, Anam

  • We did Mini Prep overnight and since I did 7 transformations, we decided to do 4 copies of each labeled A,B,C,D so that we have more flexibility in choosing our parts properly.
  • These are the following symbols we used to clarify all the tubes with the parts
    • Black circle - I 13507 (AMP Res) (A – D)
    • Red circle - F 1610 (AMP & KAN Res) (A – D)
    • Green Dot - S 01640 (AMP Res) (A – D)
    • Black dot - S 01414 (AMP Res) (A – D)
    • Red dot - J 37033 (AMP Res) (A – D)
    • Black line - J 23100 which is a promoter (AMP Res) (A – D)
    • Red line - S 01003 (AMP Res) (A – D)
  • We also labeled our colonies from the Agar plates which were chosen for miniprep overnight as it was instructed by Seema to both Anam and Esther.
  • The cells which had the promoter J 23100 were light pink colored; if Charles and Andy would like to shed some light upon that, that would be great.

August 21, 2007

Morning Session

Start Time: 11:00 am

  • Charles showed me how to use the registry properly so if anyone needs to know how to use the registry properly just let me know.
    • We searched for super parts which is more like a couple of parts ligated together so that it makes our job a lot easier
    • We chose 7 parts to be transformed from the neural network MODIFIED model
  • Part timer Muhammad Talal Latif showed up and I instructed him on how to do transformation properly
  • So I used 20 µL of TE buffer to extract:
    • J 37033: 8F, Plate:3 (Amp Res)
    • S 01640: 3L, Plate:3 (Amp Res)
  • Both of them were from the iGEM 2007 Kit Plates
  • We stored the plasmids in the –20°C freezer labeled in red DNA remaining
  • Added 4 µL of J37033 and S01640 to separate competent cell (DH5αZ1) eppendorf tubes from the –80°C freezer
  • So now we are following the Transformation steps from the guidelines posted
  • We used two AMP plates made from before
  • We also made 8 AMP plates where 4 is going to be used today for the other four parts which we also transformed:
    • I 13507: (Used form 2005 stock) (AMP Res)
    • S 01003: 20O, Plate:1 (AMP Res)
    • J 23100: 21E, Plate: 3 (AMP Res)
    • S 01414: 22O, Plate: 1 (AMP Res)
  • We also made 3 AMP and KAN plates where 1 is going to be used:
    • F 1610: 1B, Plate: 2, (AMP and KAN Resistant)
  • CONFUSION: While making the AMP and KAN plates we used AMP of 100mg/ml whereas KAN was 30mg/ml. So even though KAN was of different concentration we used the same quantity.
    • CHARLES AND ANDY GIVE US SOME INSIGHT ON TO THIS TOPIC
  • So we left all 7 plates for overnight incubation and hopefully we see some results tomorrow
  • Everybody please welcome SeungMe (part time) who came in and helped me finish the work today and of course Charles

August 13, 2007

MP and sample PD and gel run.


10 MP total:

  • 5 MP of R0040+E0240 (Black): A, B, C, D, E
  • 5 MP of R0040+E0240 (Red): A, B, C, D, E.


Sample PD was done and gel run.

Enzymes used: X/P

Three bands for all:

~3000, ~2000-2500, ~1000.

Presumably they are undigested plasmid, plasmid alone, and promoter+reporter unit.

August 12, 2007

MP O/N

5 colonies for both petri dishes of R0040+E0240.

Colonies were labelled A, B, C, D, E

Aug. 11/07 ligation dish was labelled in BLACK.

Aug 10/07 (uncertain) ligation dish was labelled in RED.


Tomorrow: MP

Note: DO NOT use the AMP in the iGEM box, as it was left out overnight at room temperature. It may have degraded (meaning, it may be unusable).

In addition:

  • Eppendorf tubes should be put into Eppendorf tube holding tray, not the test tube holding tray.
  • Also, make certain you put everything you took out of the fridge back in. Thanks.

August 11, 2007

Ligation + Transformation:

  • Ligation of R0040 and E0240 done with standard procedures.
  • Transformation of both today’s ligation and yesterday’s uncertain ligation (marked in RED) for two petri dishes total.

August 10, 2007

PD, GE, and ligation

PD and GE done with standard procedures.

Ligation could have been unsuccessful due to incubation at 37°C instead of room temperature.


Tomorrow:

  • Religate
  • Transformation

August 9, 2007

MP and sample PD run on R0040.

Tomorrow:

  • PD + Gel extraction
  • Quantitation
  • Ligation

August 8, 2007

PD of R0040. Mistake made post-gel extraction where all the purified DNA was mixed with loading dye. This mixture is in the –20 freezer at the moment.

MP O/N was prepped with R0040 A from petri dish.

Tomorrow:

Starting at 12 PM:

  1. MP (1-2 hrs), make gel while MP.
  2. make more agar (while MP)
  3. PD test run (1 hr at most)
  4. PD + gel purification/extraction (2-3 hrs)
  5. quantitation + ligation (2 hrs)

If you’re coming in tomorrow, I recommend you pack lunch.

August 7, 2007

Made stock cells of R0040 and R0011+E0240.

  • L1/A: 4 tubes
  • L1/B: 4 tubes
  • L2/A: 4 tubes
  • L2/B: 4 tubes
  • R0040: 4 tubes

They are in the –80°C freezer.

August 6, 2007

MP + Test PD

MP

  • 5 tubes:
    • R0011+E0240 L1 A,B
    • R0011 +E0240 L2 A,B
    • R0040

MP was done as usual, except for R0040, 0.5 mL was used to make stock (50/50 mix with glycerol).

PD test run

Ligated material:

  • Standard proportions used.
  • Enzymes: X/P
  • Total volume: 10mL

R0040:

  • Standard proportions.
  • Enzyme: P
  • Total volume: 10mL
  • Results:

Expected

R0040:

  • Part: 54 bp
  • Plasmid: 2079
  • Part and Plasmid: 2133

R0011:

  • Part: 55bp
  • Plasmid: 2079
  • Part and Plasmid: 2134

E0240

  • Part: 879bp
  • Plasmid: 2079
  • Part and Plasmid: 2955

R0011+E0240

  • Uncut: plasmid+promoter+gene: 3013

R0040:

  • One band b/t 2500/2000.

R0011+E0240:

  • Optimal: Two bands, 2000bp and 1000bp

Possible:

  • One cut, gene+promote+plasmid
  • Two cuts, gene+promoter, plasmid

Actual Results

  • R0040 adhered to expected results.
  • Ligated material had anomalies:
    • L1/A: 1500 bp band = ?
    • L1/B, L2/A,B: 750 bp band = ? GFP gene alone?

MP O/N done for all five.

August 5, 2007

MP O/N:

  • Five tubes
    • 1 x R0040/DH5a (A)
    • 2 x R0011+E0240 L1/(A), (B)
    • 2 x R0011+E0240 L2/(A), (B)

Standard procedures were used. The AMP used is now in the iGEM box.

Future plans:

  1. MP for all items.
  2. stock tube for E0040.
  3. If time allows:
    1. Standard PD/gel/quantitation/extraction for R0040.
    2. Test PD and gel run for ligated material
      1. Use S/P to cut out the insert; if the entire process was successful, the gel should show bands for both the R0011 and the E0240.

Making Stock:

Add same volume of glycerol as in left over MP tube. For this stock, it will be 0.5mL of MP and 0.5mL of glycerol (the glycerol is to protect the cell membranes whilst in the fridge).

August 4, 2007

Session

Did 3 transformations:

  1. E0240+R0011 into DH5aZ1, into AMP plates.
  2. E0240+R0011 into DH5aZ1, into AMP plates.
  3. R0040 into DH5a, into AMP plates.

E0240+R0011 were taken from Aug. 3, 2007 ligated stock.

R0040 directly from iGEM 2007 plate 1, well 70.

For transformations 1 and 2, standard procedures were used with small time variations.

For transformations 3:

  1. Extraction from the plate was done using 15mL of elution buffer, not deionized water.
  2. Commercial DH5α was used from Seema’s store to increase likelihood of success.
  3. Otherwise, standard procedures were used.

Further notes:

  1. We require more AMP plates. Whoever is in on Tuesday must make the plates. Remember you need to make the agar right before the autoclave cycle. Time it accordingly.
  2. Should we be starting a cloning process with DH5αZ1 if DH5α is successful? Consider uses.

Future Plans

  • Sunday: MP o/n prep.
  • Monday: MP, PD test run + gel test run
  • Tuesday: Actual PD + gel extraction + quantitate
  • Wednesday: ligation + transformation

August 3, 2007

We did three things today:

  1. Plasmid Digest
  2. Gel Run + Quantitation + Gel Extraction
  3. Ligation

Plasmid and insert (two identical copies each) were run through #1 and #2, and ligated together during #3 (final count: two tubes of ligated material).

  • Plasmid: iGEM 2006 R0011, colony ?
  • Insert: iGEM 2006 E0240, colony ? X2

As the materials used were from a successful sample PD from the day before, the entire thing was put through step #2 without a sample PD/Gel run.

Quantitation:

  • R0011: 9 ng/mL
  • E0240: 2 ng/mL

Ligation:

  • R0011 (10 ng): 1mL
  • E0240 (12 ng): 6mL
  • 10x buffer: 1 mL
  • ligase: 0.5 mL
  • ddH₂O: 1.5 mL

Plasmid, insert, and water were added first into one Eppendorf tube, then buffer and ligase. Standard procedures were followed after this point.

There were two copies of ligated material.

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