Note: I have switched with Jeremy: I will now be dealing with LuxOD47A and he will be handling LuxPQ
Purpose: To form linear copies of LuxOD47A (currently inside TOPO T/A) with BioBrick ends.
Materials and methods:
- Template: LuxOD47A in TOPO T/A [40 ng/uL]
- Forward primer: LuxOD47A BBk forward primer (Tm = 60 degrees C)
- Reverse primer: LuxOD47A BBk reverse primer (Tm = 60 degrees C)
Master Mix contents (for 15 tubes)
- 10X Pfx Amplification buffer (75 uL)
- 10mM dNTPs mixture (15 uL)
- 50mM MgSO4 (22.5 uL)
- Forward primer [10 uM] (15 uL)
- Reverse primer [10 uM] (15 uL)
- Platinum Pfx DNA polymerase [2.5 units] (10 uL)
Autoclaved distilled HOH (577.5 uL)
TOTAL: 735 uL
PCR reaction volume for Master Mix: 49 uL
PCR steps:
- Denaturation: 94 degrees C, 5 minutes
- Amplification: 36 cycles of {
- Denaturation (94 degrees C, 15 seconds);
- Annealing (58-66 degrees C, 30 seconds);
- Extension (68 degrees C, 1.5 minutes)}
- Final extension: 68 degrees C, 15 minutes
- Hold temperature: 4 degrees C
12 tubes were prepared to span the gradient, each of which contained 1 uL of template DNA and 49 uL master mix. A negative control with ddHOH in lieu of template DNA was also prepared.
Results:
The gradient PCR products were visualised on a 1% agarose gel, as shown below. The right-most lane is the negative control, although it can’t really be distinguished in the results. This doesn’t seem to have worked, so it will be repeated later.
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