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From 2011.igem.org

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	===July, Week 2===		===July, Week 2===
-	====7-13-2011====
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-	Retrieved gel from cold room and covered with 1x TAE in gel box and heated it (by running the gel with nothing in it)
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-	Prepared Mutagenesis PCR products from last Thursday to be run on Gel
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-	The tubes I made up came from the following:
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-	A = Green/ Blue tubes conatained Dr. Burkett's dNTP's
-	B = Orange tubes contain Dr. Goode's dNTP's
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-	I prepared 8 tubes as follows:
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-	CA  = Pink tube contains 10 microliters of the Control A sample and 2 microliters of loading dye
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-	CB  = Blue tube contains 10 microliters of the Control B sample and 2 microliters of loading dye
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-	Rxn 1 A & B = Pink A tube contains 10 microliters of the RXN 1 A sample and 2 microliters of loading dye
-	Blue B tube contains 10 microliters of the RXN 1 B sample and 2 microliters of loading dye
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-	Rxn 4 A & B = Pink A tube contains 10 microliters of the RXN 4 A sample and 2 microliters of loading dye
-	Blue B tube contains 10 microliters of the RXN 4 B sample and 2 microliters of loading dye
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-	Rxn 5 A & B = Pink A tube contains 10 microliters of the RXN 5 A sample and 2 microliters of loading dye
-	Blue B tube contains 10 microliters of the RXN 5 B sample and 2 microliters of loading dye
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-	These 8 tubs are located in the freezer and are ready to be run on the gel. They are in a yellow rack with a pink taped labeled with todays date and iGEM, mutagenesis PCR for gel.
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-	Dr. Goode requested that the gel lanes be filled as follows
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-	1.  Ladder
-	2.  CA
-	3.  CB
-	4.  R1A
-	5.  R1B
-	6.  Ladder
-	7.  R4A
-	8.  R4B
-	9.  R5A
-	10. R5B
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-	--[[User:Mduley|Mduley]] 18:00, 13 July 2011 (CDT)

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Revision as of 17:39, 14 July 2011

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Contents 1 Notebook 1.1 Calendar 1.1.1 July 1.2 July, Week 2

Notebook

Run through for abstract and scheduling:

Start with plasmid with taq gene and pst1 gene

Remove pst1 site from taq coding sequence

• In order to remove the restriction site, do site directed mutagenesis (1day)
• PCR
• Digestion
• Transformation
• Screening (1day)
  • Dilute DNA
  • Colony PCR individually
  • Digestion of PCR product with pst1
  • Run gel ~2500bp

Add biobrick prefix/suffix to taq coding sequence

• PCR and add the prefix and suffix as primers (one prefix has an extra AG-make sure to use the correct one) (1-2 days)
  • Run gel
  • Cut out of gel
• Cut with restriction enzyme
• Clean up DNA
• Ligation with vector (could be vector with the terminator sequence, promoter and RBS)
• Transformation

Add a promoter, transcriptional terminator, ribosome binding site (RBS)

• Screen colonies (1 day)
  • Colony PCR
  • Restriction Digestion
  • Clean up DNA
• Sequence (1 day)

Make taq protein

Compare it to other enzymes and make sure it works

Calendar

July

Week 2: Tuesday, July 12; Wednesday, July 13; Thursday, July 14; Friday, July 15; Saturday, July 16; Sunday, July 17;

Week 3: Tuesday, July 19; Wednesday, July 20; Thursday, July 21; Friday, July 22; Saturday, July 23; Sunday, July 24;

July, Week 2