Team:UCLA/Notebook/Spider Silk Genetics/27 August 2015

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8/27/2015

Sequencing Results

• M1-12(1C3)
  • 1: wrong sequence entirely
  • 2: wrong sequence entirely
  • 3: wrong sequence entirely
  • Interestingly, all three sequences align to each other.
• M1/2[1:1]-12(1C3)
  • 1: Bad, single base pair mutation at ~550
  • 2: GOOD
  • 3: GOOD
• M1/2[1:1]-12(T7)
  • 1: Bad, deletion at ~80
  • 2: GOOD
  • 3: possibly correct, but not trustworthy due to short read.
• M1/2[2:1]-12(1C3)
  • 1: Bad, single bp mutation near ~660
  • 2: Bad, single bp mutation at 962
  • 3: GOOD
• M1/2[2:1]-12(T7)
  • 1: bad, non-silent single bp mutation at 1004
  • 2: GOOD? silent single bp mutation at 702
  • 3: GOOD? silent single bp mutation at 702

Miniprep for M1/2[1:2]-12(1C3)

• Used qiagen kit to purify the two cultures.

Digestion Check for M1/2[1:2]-12(1C3)

• Digest with X, P in preparation for possible subcloning.
• Digest 3 ug of each DNA in a 50 uL reaction with 1 uL each of XbaI, PstI.
• Digest at 37 C for 1.5 hrs, heat kill at 65 C for 20 min.