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Revision as of 02:57, 27 October 2007 (view source) Jmonk (Talk | contribs) (→Bio-Brick Creation) ← Older edit		Revision as of 02:59, 27 October 2007 (view source) Jmonk (Talk | contribs) (→Bio-Brick Creation) Newer edit →
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	[[Digestion with Restriction Enzymes]]		[[Digestion with Restriction Enzymes]]
-	PCR purification	+	[[PCR purification]]
-	Ligation	+	[[Ligation]]
-	Transform and pick colonies	+	[[Transform and pick colonies]]
-	Miniprep and determine concentrations	+	[[Miniprep and determine concentrations]]
-	Restriction Map	+	[[Restriction Map]]
-	Retransform with chosen plasmid	+	[[Retransform with chosen plasmid]]
-	Maxiprep and determine concentraions	+	[[Maxiprep and determine concentraions]]
	Sequence		Sequence

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Revision as of 02:59, 27 October 2007

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.

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

Digestion with Restriction Enzymes

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

Bio-Brick Verification: Quality Control

Grow cells to confluence

Seed cells

Infect cells with virus

Change media next day

Cellular experiments

- Microscopy

- FACS