Team:Illinois/Notebook

From 2009.igem.org

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*0.4 μL BSA
*0.4 μL BSA
*3.6 &mu;L dH<sub>2</sub>O
*3.6 &mu;L dH<sub>2</sub>O
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== '''September 28''' ==
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PCRed the Lac promoter, the Tet promoter (BBa_R0040), and the Tet promoter-RBS construct in a 100 &mu;L reaction using
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*2 &mu;L template
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*0.8 &mu;L each primer
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*20 &mu;L 5x Phusion HF Buffer
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*2 &mu;L dNTP mix
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*0.6 &mu;L Phusion polymerase
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*73.8 &mu;L dH<sub>2</sub>O
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 +
Also performed dpnI digestion (3 &mu;L) for 4.75 hours.
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== '''September 29''' ==
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PCRed sRNA genes DicF, gcvB, and SgrS out of the E. coli chromosome in 50 &mu;L reactions using
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*4 &mu;L template
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*0.4 &mu;L each primer
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*5 &mu;L 10x buffer
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*1 &mu;L dNTP mix
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*0.8 &mu;L Pfu polymerase
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*38.4 &mu;L dH<sub>2</sub>O
 +
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== '''September 30''' ==
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 +
Digested the Lac promoter, Tet promoter, and Tet promoter-RBS construct from the September 28 PCR for four hours using
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*0.4 &mu;L PstI
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*30 &mu;L DNA
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*3.5 &mu;L BSA
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*0.5 &mu;L dH<sub>2</sub>O
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Also ligated and transformed Vogel sRNAs, but did not see any colonies.
 +
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Revision as of 21:49, 19 October 2009

Click to go to the Illinois home page




Contents

Notebook

This page will include periodic entries during our research. We will be documenting the progression of our project, any problems or obstacles encountered, and solutions to problems.

Browse by Lab Team

Click on a group name below to view notebook entries and information pertaining to one of our individual lab teams.

Post-Summer Progress

This section includes entries for lab work done after the summer was over.

September 24

PCRed all target sequences w/ BB sRNAs w/ no results

September 25

PCRed sRNA genes (MicC, MicA, MicF) and their respective target sequences (OmpC, OmpA, OmpF). The gel was run with the Vogel primers as a control since the previous attempt to PCR Biobricks failed.

Also PCRed plasmid pSB1A2. Used 56°C for annealing temperature instead of 58°C and got better results.

Also restreaked plates of E. coli containing plasmid PJU-334 and Biobricks BBa_K091101, BBa_I0500, and BBa_R0040.

September 26

PCRed sRNA target sequences OmpC, OmpA, OmpF, FtsZ, PtsG, GalK,. For PCRs, ran a 50 μL reaction using

  • 4 μL chromosomal template
  • 0.4 μL of each primer
  • 5 μL 10x buffer
  • 0.8 μL Pfu polymerase
  • 1 μL dNTP mix
  • 38.4 μL dH2O

Also digested OmpC, OmpA, and OmpF target sequences from yesterday's PCR. The first round of digestions was run for three hours using

  • 1 μL EcoRI-HF (20,000 U/mL)
  • 3.5 μL 10x Buffer 4
  • 30 μL DNA
  • 0.35 μL BSA
  • 0.15 μL dH2O

The second round of digestions used

  • 0.2 μL PstI (100,000 U/mL)
  • 3.5 μL 10x Buffer 3
  • 30 μL DNA
  • 0.35 μL BSA
  • 1 μL dH2O

Also cultured cells containing plasmid PJU-334 and Biobricks BBa_K091101, BBa_I0500, and BBa_R0040 overnight.


September 27

Miniprepped cells containing plasmid PJU-334 and Biobricks BBa_K091101, BBa_I0500, and BBa_R0040. Also digested target sequences from yesterday. For the first round of digestions, ran for three hours using

  • 0.4 μL PstI (100,000 U/mL)
  • 3.5 μL 10x Buffer 3
  • 30 μL DNA
  • 0.35 μL BSA
  • dH2O to 3 μL

For the first round of digestion, the incorrect buffer may have been used. The digestion was then cleaned up by PCR cleanup, and then the second round of digestions were run for 2.5 hours using

  • 2 μL EcoRI-HF (20,000 U/mL)
  • 4 μL 10x Buffer 4
  • 30 μL DNA
  • 0.4 μL BSA
  • 3.6 μL dH2O

September 28

PCRed the Lac promoter, the Tet promoter (BBa_R0040), and the Tet promoter-RBS construct in a 100 μL reaction using

  • 2 μL template
  • 0.8 μL each primer
  • 20 μL 5x Phusion HF Buffer
  • 2 μL dNTP mix
  • 0.6 μL Phusion polymerase
  • 73.8 μL dH2O

Also performed dpnI digestion (3 μL) for 4.75 hours.

September 29

PCRed sRNA genes DicF, gcvB, and SgrS out of the E. coli chromosome in 50 μL reactions using

  • 4 μL template
  • 0.4 μL each primer
  • 5 μL 10x buffer
  • 1 μL dNTP mix
  • 0.8 μL Pfu polymerase
  • 38.4 μL dH2O

September 30

Digested the Lac promoter, Tet promoter, and Tet promoter-RBS construct from the September 28 PCR for four hours using

  • 0.4 μL PstI
  • 30 μL DNA
  • 3.5 μL BSA
  • 0.5 μL dH2O

Also ligated and transformed Vogel sRNAs, but did not see any colonies.

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