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August 9, 2007

  • Ran PCR cleanup on the 8 PCR rxns that worked from yesterday (all except 0.1uL for Buffer#1). Combined all into one sample.
  • Restriction digest with EcoR1 and SpeI
  • Assumed we maxed out the spin column so 10ug DNA was eluded in 28uL EB Buffer leading to a DNA concentration of ~357ng/uL

Restriction Digest Protocol

1. Added to tubes:

  • 2uL DNA (~350 ng/uL)
  • 1uL 10x Buffer
  • 1uL BSA
  • 0.5uL each restriction enzyme
  • 5uL dH2O

2. Gingerly flick tube to mix 3. Spin down tubes briefly 4. Incubate at 37C in water bath for 1-4 hrs (1-2 hrs optimal)

  • Also doing restriction on P1010 w/ psB1A2 which will be the vector for the following ligation.
  • Started incubating at 12:45pm
  • Ran P1010 on a gel to isolate it from psB1A2
  • Seems the restriction didnt work as planned. Going to grow up P1010 in psB1A2 overnight and miniprep and restrict again in the morning.


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