Continuing colony PCR of putative clones
Based on the strange results obtained/analyzed between Saturday/Monday, I'm going to do some more definitive tests on the streakouts.
- 50-68°C gradient on gDNA and #31 (different patterns) with new upF/dnR primers (+:5.5 kb, -:nothing or nonspecific)
- This should find me a good temperature for amplifying the whole fragment, and will let me know that the accBCDA insert is in the cell. I should be able to assert that growth means acsA has been knocked out as well, though there is potential for spontaneous loss of function.
- accBCDA-F + old dnR primer (+:5 kb, -:nothing)
- This will actually tell me that the accBCDA insert is where it should be. Hopefully has a better chance of working than the original upF/dnR primers, which are trouble.
Whole insert, new primers, temperature gradient
- Just doing #31 and the negative control, they had significantly different bands before
- 8 of each, between 50 and 68 degrees
10 μL reaction:
- 5 μL GoTaq (85 in master mix)
- 2 μL H2O (34)
- 1 μL new acsA-up-F primer (17)
- 1 μL new acsA-down-R primer (17)
- 1 μL template (dilute 7002 gDNA or resuspended #31 in water)
Thermal cycler program:
- 98°C 2:00
- 98°C :30
- 50-68°C :30
- 72°C 5:00 (5 kb with GoTaq)
- Goto 2 34x
- 72°C 10:00
accBCDA to chromosome
- 23 colonies, picked from what's available and struck out in patches on a 500 μM plate
10 μL reactions:
- 5 μL GoTaq (125 in master mix)
- 3 μL H2O (75)
- 1 μL accBCDA-F primer (25)
- 1 μL original acsA-down-R primer (25)
Thermal cycler program:
- 98°C 2:00
- 98°C :30
- 54°C :30 (according to NEB calculator with Taq polymerase, 100 μM primers)
- 72°C 5:45 (5.5 kb with GoTaq)
- Goto 2 34x
- 72°C 10:00
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Cyanobacterial acrylate production
