User:Eric M. Walters/Notebook/Spring 2012/2012/06/12

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Beginning pheA construction

  • pheA-up amplified with 69 annealing (.5 kb)
  • pheA-dn with 70 (.5 kb)
  • acsA-kanR construct with 66 (3.3 kb)


pheA digests

  • pheA-up gets speI
  • pheA-dn gets sphI
  • acsA-kanR gets both

All will be 50 uL, run for an hour at 37, then heat inactivated at 80 (20 minutes), then held during meeting to be ligated O/N afterward.

  • 30 uL DNA
  • 5 uL Buffer 4
  • 5 uL BSA
  • 1-2 uL enzyme
  • 9-8 uL nuclease free water