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Contents 1 Notebook 1.1 Calendar 1.1.1 July 1.2 July, Week 2 1.2.1 7-13-2011

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

7-13-2011

Retrieved gel from cold room and covered with 1x TAE in gel box and heated it (by running the gel with nothing in it)

Prepared Mutagenesis PCR products from last Thursday to be run on Gel

The tubes I made up came from the following:

A = Green/ Blue tubes conatained Dr. Burkett's dNTP's B = Orange tubes contain Dr. Goode's dNTP's

I prepared 8 tubes as follows:

CA = Pink tube contains 10 microliters of the Control A sample and 2 microliters of loading dye

CB = Blue tube contains 10 microliters of the Control B sample and 2 microliters of loading dye

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

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

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

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.

Dr. Goode requested that the gel lanes be filled as follows

1. Ladder 2. CA 3. CB 4. R1A 5. R1B 6. Ladder 7. R4A 8. R4B 9. R5A 10. R5B

--Mduley 18:00, 13 July 2011 (CDT)