From 2007.igem.org

Overview || People || Lab work || Experiments || Bioinformatics || Simulations

Contents 1 Experimentation 1.1 Protocols 2 Assembly Line Flow 2.1 Bio-Brick Creation 2.2 Bio-Brick Verification: Virus Harvest 2.3 Bio-Brick Verification: Quality Control

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

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