User:Eric M. Walters/Notebook/Spring 2012/2012/05/22

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Digestion

  • Cleaned up the PCR amplifications from yesterday, ran a gel of some leftover non-cleaned DNA
    • 5-22.png
  • Nanodrop DNA concentrations
    • acsA-up BC: 87 ng/μL
    • acsA-dn BC: 93 ng/μL

SphI-HF was used to digest, in Buffer 4 at 37°C, 45 minutes (10:45-11:30). Heat inactivated at 65°C for 20 minutes.

  • 30 μL DNA
  • 5 μL Buffer 4
  • 1 μL SphI-HF
  • 14 μL H2O

Ligation

Ligation will be done for 30 minutes at room temperature (12:05-12:35), per Matt's advice. 50 μL, since that's what I've needed before.

  • 5 μL ligase buffer
  • 2.5 μL T4 DNA ligase (1 per 20, from NEB)
  • 15 μL downstream fragments
  • 15 μL upstream fragment
  • 12.5 μL H2O

Gel after 30 minutes (but continued for another 60):

Pflegerlab 2012-05-22 13hr 29min acsA up+dn BC ligation.jpg


Amplification

I want to increase the amount of ligation produce I've got to transform with via PCR. 2x50 μL reactions

Per 50 μL:

  • 35 μL H2O
  • 10 μL Buffer HF
  • 1 μL dNTPs
  • 1 μL acsA-upFv2
  • 1 μL acsA-dnRv2
  • 1 μL heat-inactivated ligation product
  • 1 μL Phusion

Run overnight to be cleaned, AGEd, and transformed.