Princeton/lab/experimentation
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| + | <img src="http://img341.imageshack.us/img341/8195/logo5oz0.jpg" alt="LOGO"> | ||
| + | </html> [[Princeton/overview | Overview]] || [[Princeton/People | People ]] || [[Princeton/lab | Lab work]] || [[Princeton/lab/experimentation | Experiments]] || [[Princeton/lab/bioinformatics | Bioinformatics]] || [[Princeton/lab/simulation | Simulations]] | ||
=Experimentation= | =Experimentation= | ||
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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. | 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. | ||
| - | + | =Assembly Line Flow= | |
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Design Primers | Design Primers | ||
| - | PCR | + | [[PCR]] |
| + | |||
| + | [[Run Gel]] | ||
| + | |||
| + | - Check Primer concentration | ||
| + | |||
| + | - Check Primer design | ||
| + | |||
| + | Gel Extraction | ||
| + | |||
| + | [[Digestion with Restriction Enzymes]] | ||
| + | |||
| + | [[CIP Treatment]] | ||
| + | |||
| + | [[PCR purification]] | ||
| + | |||
| + | [[Ligation]] | ||
| + | |||
| + | [[Transform and pick colonies]] | ||
| + | |||
| + | [[Miniprep and determine concentrations]] | ||
| + | |||
| + | [[Restriction Map]] | ||
| + | |||
| + | [[Retransform with chosen plasmid]] | ||
| + | |||
| + | [[Maxiprep and determine concentraions]] | ||
| + | |||
| + | Sequence | ||
| + | |||
| + | == Bio-Brick Verification: Virus Harvest == | ||
| + | |||
| + | Prepare cell culture media | ||
| + | |||
| + | Seed cells | ||
| + | |||
| + | Check health of cells | ||
| + | |||
| + | Transfect | ||
| + | |||
| + | Check phenotype 24hrs post transfection | ||
| + | |||
| + | Transform correct try using XL-10 cells | ||
| + | |||
| + | Maxiprep and ethanal purify the try and packaging plasmids | ||
| + | |||
| + | Seed 293FT cells | ||
| + | |||
| + | Check health of cells 24hrs later | ||
| + | |||
| + | Transform the vector and packaging plasmids using the CaPO4 method | ||
| + | |||
| + | Check for fluorescence after 24hrs | ||
| + | |||
| + | Harvest virus and concentrate using ultra-centrifugation | ||
| + | |||
| + | Please see [[Making A Lentivirus]] for protocols | ||
| + | |||
| + | == Bio-Brick Verification: Quality Control == | ||
| + | |||
| + | Grow cells to confluence | ||
| + | |||
| + | Seed cells | ||
| - | + | Infect cells with virus | |
| - | + | Change media next day | |
| - | + | ||
| - | + | Cellular experiments | |
| - | + | - Microscopy | |
| - | + | - FACS | |
| - | + | Please see [[Making A Lentivirus]] for protocols | |
Latest revision as of 04:00, 27 October 2007
Overview || People || Lab work || Experiments || Bioinformatics || Simulations
Contents |
Experimentation
Protocols
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.
Assembly Line Flow
Bio-Brick Creation
Design Primers
- Check Primer concentration
- Check Primer design
Gel Extraction
Digestion with Restriction Enzymes
Miniprep and determine concentrations
Retransform with chosen plasmid
Maxiprep and determine concentraions
Sequence
Bio-Brick Verification: Virus Harvest
Prepare cell culture media
Seed cells
Check health of cells
Transfect
Check phenotype 24hrs post transfection
Transform correct try using XL-10 cells
Maxiprep and ethanal purify the try and packaging plasmids
Seed 293FT cells
Check health of cells 24hrs later
Transform the vector and packaging plasmids using the CaPO4 method
Check for fluorescence after 24hrs
Harvest virus and concentrate using ultra-centrifugation
Please see Making A Lentivirus for protocols
Bio-Brick Verification: Quality Control
Grow cells to confluence
Seed cells
Infect cells with virus
Change media next day
Cellular experiments
- Microscopy
- FACS
Please see Making A Lentivirus for protocols
