Team Fence
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
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
- Ladder
- Mira N SS
- Mira C SS
- Brazz N SS
- Brazz C SS
- V24 N/S
- B21 X/P
- Ladder
- Gel bands indicated were cut and purified from the gel.
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