IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/08/02: Difference between revisions

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[[Image:VP16PCR.2_blank_8-2.jpg]]
[[Image:VP16PCR.2_blank_8-2.jpg]]
===VP16 PCR.3===
60μL reaction:
*1.5 μL pfx polymerase
*3μL DNTPs
*18μL enhancer buffer
*1.5μL MgSO<sub>4</sub>
*3μL 1/10 dilution VP16.Hind.Fwd
*3μL 1/10 dilution VP16.Rev
*3μL 1/10 dilution of VP16 (1)
*12μL amp buffer
*5μL DH<sub>2</sub>O
Using modified program:
  1=94°C for 2:00
  2=94°C for :15
  3=60°C for :30
  4=70°C for :45
  5=Goto 2, 30 times
  6=4.0°C forever
  7=end
(see tomorrow's entry for gel image)


===Ligations===
===Ligations===

Revision as of 13:43, 7 September 2010

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Team Fence

Gel extraction

Gal4 spe bam 8/2 and EcR bam xba 8/2 gel extracted and purified.

Gal4 on the left, EcR on the right


Nanodrop of Gal4 and EcR Gel Extractions

Plasmid Quantity (ng/μL) 260/280
Gal4 (1) 1.4 -3.18
Gal4 (2) 7.2 4.59
EcR (1) 5.3 2.52
EcR (2) 5.6 2.12

VP16 PCR 2

60μL reaction:

  • 1.5 μL pfx polymerase
  • 3μL DNTPs
  • 18μL enhancer buffer
  • 1.5μL MgSO4
  • 3μL 1/10 dilution VP16.Hind.Fwd
  • 3μL 1/10 dilution VP16.Rev
  • 3μL 1/10 dilution of VP16 (1)
  • 12μL amp buffer
  • 5μL DH2O

using program: 

  1=94°C for 2:00
  2=94°C for :15
  3=68°C for :30
  4=Goto 2, 30 times
  5=4.0°C forever
  6=end


No PCR product is visible

VP16 PCR.3

60μL reaction:

  • 1.5 μL pfx polymerase
  • 3μL DNTPs
  • 18μL enhancer buffer
  • 1.5μL MgSO4
  • 3μL 1/10 dilution VP16.Hind.Fwd
  • 3μL 1/10 dilution VP16.Rev
  • 3μL 1/10 dilution of VP16 (1)
  • 12μL amp buffer
  • 5μL DH2O

Using modified program: 

  1=94°C for 2:00
  2=94°C for :15
  3=60°C for :30
  4=70°C for :45
  5=Goto 2, 30 times
  6=4.0°C forever
  7=end

(see tomorrow's entry for gel image)

Ligations

  Gal4 into EcR
  Barnase into B15
  LTP into B11 (positive control from team allergy)

Gal4

  • 3μL Gal4 spe bam gel pur. (2) 8/2
  • 9μL EcR xba bam gel pur. (2) 8/2
  • 2μL 10x T4 ligase buffer
  • 5μL DH2O
  • 1μL T4 ligase

EcR control

  • 9μL EcR xba bam gel pur. (2) 8/2
  • 2μL 10x T4 ligase buffer
  • 8μL DH2O
  • 1μL T4 ligase

Barnase

  • 1.5μL Barnase digest gel pur. (1) 7/29
  • 3μL B15 spe pst phosphorylated clean up
  • 2μL 10x T4 ligase buffer
  • 12.5μL DH2O
  • 1μL T4 ligase

B15 control

  • 3μL B15 spe pst phosphorylated clean up
  • 2μL 10x T4 ligase buffer
  • 14μL DH2O
  • 1μL T4 ligase

LTP

  • 2μL LTP
  • 1μL B11 backbone
  • 2μL 10x T4 ligase buffer
  • 16μL DH2O (should have been 14μL, the 16 was accidental)
  • 1μL T4 ligase

B11 control

  • 1μL B11 backbone
  • 2μL 10x T4 ligase buffer
  • 16μL DH2O (should have been 14μL, the 16 was accidental)
  • 1μL T4 ligase

Allowed tubes to sit at room temp for an hour, proceeded to transformation

Team Flavor

DTL of Mira/Brazz+StrepII+Stop into V24

  • Digested 1 microgram of DNA for each insert and for V24 backbone.
    • Digested inserts and backbone with NotI/SpeI
  • Gel extracted and purified bands indicated in image below
  • Ligated insert into 50ng of V24 backbone with a 3:1 insert to backbone ratio
  • Transformed and plated on LB+AMP plates and left in 37°C incubator overnight

DTL of Wintergreen pathway

  • Digested 400ng of J45004, 20ng of J45017, and 1 microgram of B21 (for v0120 backbone)
    • Digested J45004, J45017, and B21 BB with XbaI/PstI
  • PCR purified digested inserts
  • Gel extracted and purified B21 BB (see gel image below)
  • Ligated inserts into 50ng of B21 v0120 backbone with a 3:1 insert to backbone ratio
    • Transformed and plated on LB+AMP plates and left in 37°C incubator overnight

Gel Purification

  1. Ladder
  2. Mira N SS
  3. Mira C SS
  4. Brazz N SS
  5. Brazz C SS
  6. V24 N/S
  7. B21 X/P
  8. Ladder
  • Gel bands indicated were cut and purified from the gel.


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