IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/08/02

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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



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

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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