Team:UCLA/Notebook/Spider Silk Genetics/2 September 2015

iGEM UCLA




9/2/2015

Sequencing Results

  • M1-12(1C3)
    • 1: GOOD
    • 2: Bad, deletion in suffix
    • 3: GOOD
  • M1-12(T7)
    • 1: incorrect sequence
    • 2: GOOD
    • 3: Bad, no reverse priming
  • M1/2[1:2]-12(1C3)
    • 1: GOOD
    • 2: GOOD
    • 3: GOOD
  • M1/2[1:2]-12(T7)
    • 1: Bad, no priming
    • 2: Bad, non-specific
    • 3: GOOD

Gel Purification for M1-15

  • Used zymo kit.
  • Yield 31.64 ng/uL A: 2.47
  • Need to do another PCR to get more.

PCR Amplification for M1-15

  • Used 1:1000 dilution of M1-15 from above as template.
    • Used ~100 pg of template in reaction.
  • Set up 2x 50 uL reactions
Volume (uL)
5x Q5 Buffer 10
10 mM dNTPs 1
10 uM For (F-03) 2.5
10 uM Rev (G-03) 2.5
Template (~30 pg/uL) 3j
Q5 Polymerase 0.5
ddH2O 30.5
Total 50
98 C 30 sec
98 C 10 sec
66 C 20 sec
72 C 40 sec
repeat from step 2 25x
72 C 2 min
12 C hold
  • Verified 5 uL of each reaction on 1% TAE gel with 2 uL of 1 kb ladder.
Fig. 1 PCR amplification of M1-15 using previous M1-15 as template. The expected band size is 1630 bp.
  • Decided that the band looks clean enough, so will PCR purify and not gel purify.
    • Eluted in 12 uL.

Digestion for M1-15

  • Digest 5 uL of the PCR product with E,P and 5 uL of PCR product with X,P.
  • Digest in a 50 uL reaction with buffer 2.1 and 1 uL of each relevant enzyme.
  • Digest at 37 C for 1.5 hrs, heat kill at 65 C for 20 min.
  • PCR purified the digestion product.

Ligation for M1-15

  • Ligate E,P digested products into pSB1C3.
  • Ligate X,P digested products into BBa_K525998(S,P)
  • Ligate at a 1:3 molar vector to insert ratio.
  • Ligate at 25 C for 1 hr, heat kill at 65 C for 20 min.

Transformation

  • Transform M1-15(1C3) into DH5(alpha) electrocompetent cells.
    • Dialyze against ultra-pure ddH2O.
    • Tried 3 times. First two times, the sample arced. Third time transformed 2 uL of a a 1:50 idlution.
    • Arc timme 5.4 s.
  • Transform M1-15(T7) into BL21(DE3) chemically competent cells.