Morning: Digest of 3 PCR products
- Time limited changes: didn't run gel of products first, will digest for 3 hours (BamHI can't be heat inactivated)
- Concentrations (digested volume DNA/volume water in 50 μL digest)
- Up: 112 (36/8)
- Down: 111 (16/28)
- Acc: 190 (22/21)
Afternoon: gel extraction and ligation
- Ran gel of uncut products
- Expected sizes present, BUT accBCDA also had a concentrated, smaller band (a first)
- Gel extracted all 3
- Good yields, up: 82 ng/μL, down: 42 ng/μL, accBCDA: 49 ng/μL
- 50 μL ligation in 1:3:1::up:acc:down mass ratio (3:1:3 molar)
- 5 μL ligase buffer
- 5 μL T4 DNA ligase
- 3.5 μL acsA-up (E cut)
- 7 μL acsA-down (B cut)
- 21 μL accBCDA (EB cut)
- 8.5 μL H2O
16°C overnight for verification, gel extraction, re-amplification (with v2 primers) tomorrow
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Cyanobacterial acrylate production
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