User:Eric M. Walters/Notebook/Spring 2012/2012/02/08

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Cleaning, digesting, and ligating the up-acc-dn construct

PCR products

  • Correct sizes, post cleanup nanodrop values:
    • acsA-up: 103 ng/ul
    • acsA-dn: 61 ng/ul
    • accBCDA: 170 ng/ul

Digests

  • All digests get 5 ul buffer 4, 30 ul of the PCR product, and water up to 50 ul.
  • acsA-up gets EcoRI(hf)
  • acsA-dn gets BamHI(hf)
  • accBCDA gets EcoRI and BamHI

Run 4:15 - 6:30

  • Bands for up and down looked fine, accBCDA had a bit of a smear around the band

Ligation

1 ul up 1 ul down 6 ul accBCDA 1 ul buffer 1 ul ligase