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Ethanol
Got plasmid PACYC Duet-pdc-adhb in strain DH10b and strain BL21 from Nielsen's lab to use for ethanol production
- Plated both in preparation to transform plasmid into BL21 Strain.
Transformed ligation mixture
- Ligation mixture
- - Control
- + Control (ACC Plasmid)
PCR Amplification of ACC parts
First, the dry primers were made into 100 μM master stocks. 1 μL was added per .1 nm. For example, a 31.2 nm sample had 312 μL added. This master stock was then diluted into working stocks of 5 μM. This was done by adding 1 μL of master mix into 19 μL H2O. This was done for all eight primers , four forward and four reverse.
The ACC plasmid was then diluted from 54.2 μg/μL to about 1 μg/μL through a 1/50 dilution.
PCR mix for all four reactions (accA, accB, accC, and accD)
- PCR reaction
Reagent
|
|
2x GoTaq Master Mix |
12.5
|
Forward Primer (working stock) |
1.0
|
Reverse Primer (working stock) |
1.0
|
Template DNA (Diluted) |
1.0
|
H2O |
9.5
|
Total |
25.0
|
Initial denature step of 95 °C for 2 min
|
PCR cycle: 95 °C for 30 sec -> 45 °C for 45 sec -> 73 °C for 80 sec
|
Final annealing step at 73 °C for 5 min
|
|