User:Eric M. Walters/Notebook/Spring 2012/2012/04/10

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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