Screening putative acsA::accBCDA clones
- Many small colonies arose from the streakouts done Monday of the two transformant colonies
- Will be screening both of them with the original (now outside of the insertion) primers acsA-upF and -dnR
- Controls are gDNA (should yield just the acsA + 2x500 bp flanking regions - 3.2 kb total), and a ΔacsA 7002 gDNA tube I found in Matt's freezer. He's not in right now, but this should let me make sure that the process of deleting acsA hasn't disrupted the near-outside primers I'm using. However, I don't yet know what size I'm expecting to see.
Colony PCR reactions and conditions
- 20 μL reactions:
- 10 μL GoTaq
- 1 μL acsA-upF
- 1 μL acsA-dnR
- Either 1 μL gDNA, or pick of colony
- 7 μL H2O
- PCR cycle:
- 95°C - 2:00
- 95°C - 0:30
- 61°C - 0:30
- 72°C - 5:40 (5.6 kb at 1 kb/min)
- Repeat 30x
- 72°C - 5:00
Gel after 45 minutes at 100V:
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Cyanobacterial acrylate production
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