Princeton/lab/experimentation
From 2007.igem.org
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.
Please consult the relevant vendors for protocol documents.
Assembly Line Flow
Bio-Brick Creation
Design Primers
PCR
Run Gel
- Check Primer concentration - Check Primer design
Gel Extraction and determine concentrations
Digestion with Restriction Enzymes
PCR purification and determine concentrations
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
Bio-Brick Verification: Quality Control
Grow cells to confluence
Seed cells
Infect cells with virus
Change media next day
Cellular experiments
- Microscopy - FACS
