Team:UCLA/Notebook/Spider Silk Genetics/19 August 2015

iGEM UCLA




8/19/2015

Sequencing Results

  • M1-12(1C3):
    • 1: BAD (VR is bad; no priming)
    • 2: good
    • 3: good
    • 4: good?
      • Further inspection reveals BBa_K525998 T7-RBS is present in the beginning. The sequence is otherwise correct. This is suspicious, and we will do a Restriction test to verify proper length.
  • ERRATA: All these sequences, while correct, are not the appropriate size. These are in fact, M1-9(1C3) and M1-9(T7) plasmids.

ICA for M1/2[1:1]-12

  • Followed previous ICA protocol using:
    • 45 minute initiator binding time.
    • 3 minute ligation times for each step.
    • Elute in 15 uL of 0.01% Tween-20 at 95 C for 5 min.

PCR Amplification for M1/2[1:1]-12

  • 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 (ICA eluate) 1
ddH2O 32.5
Q5 Polymerase 0.5
Total 50
98 C 30 sec
98 C 10 sec
66 C 20 sec
72 C 30 sec
repeat from step 2 20x
72 C 2 min
12 C hold

Results

  • Cast 1% TAE gel to visualize. Used 2 uL of NEB 1 kb ladder.
Fig. 1PCR amplification of M1/2[1:1]-12 ICA eluate. Both lanes are identical reactions. The expected size for 12-mer amplification is 1324 bp. The most prominent band in both reactions is a ~1.3 kb band. There are accessory bands corresponding to 3-, 6-, 9-mers as well.
  • This result indicates that ICA for M1/2[1:1]-12 was successful.

Large Scale Liquid Culture for M2-AB, BC, CA

  • 100 mL of each. Picked a single colony from freshly streaked plates. Used the old version of M2 monomers for this.