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

iGEM UCLA




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.
  • Visualize on 1% gel with 2 uL of NEB 1 kb ladder.
Fig. 1 X, P digest of putative M1/2[1:2]-12(1C3). The expected sized bands are 2070 and 1324, corresponding to the vector and the insert respectively. Neither colony has the correct sized band at 1324 bp.
  • Both colonies were wrong; did not send for sequencing.

BsaI Digestion of M1 Monomers

  • Digested 5 ug of plasmid DNA in a 50 uL reaction with 4 uL of BsaI. Digest 2x 50 uL reactions for each monomer.
  • Digest at 50 C for 2 hrs, heat at 65 C for 20 min.
  • Run of 1.5% TAE gel to visualize. Used 2 uL of NEB 1 kb ladder. (Should have used the 50 bp or 100 bp ladder instead)
Fig. 2 BsaI Digestion of M1 monomers. The expected size for digested monomer is 102 bp.
  • Olivia excised the gel for extraction.