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

iGEM UCLA




7/27/2015

Transformation Results

  • 5 colonies on the M2-12(1C3) plate

Colony PCR

  • For M2-12(1C3) colonies
  • Resuspend colony in 50 uL LB.
  • Use 1 uL as a template in 2x Taq Red PCR.
Volume (uL)
2x Taq Red 12.5
10 uM For (VF) 1.25
10 uM Rev (VR) 1.25
Template (1:50) 1 uL
ddH2O 9 uL
Total 25 uL
95 C 3 min
95 C 25 sec
56 C 30 sec
72 C 45 sec
repeat from step 2 30x
72 C 5 min
12 C hold

PCR Amplification of 15-mer

  • Set up 2x 50 uL Reactions for amplification using M2-15mer template.
  • Scaled up reactions from 7/24/2015.
  • Same conditions, but with 23 cycles instead of 20 cycles.
  • Proceed to gel purification.

Results

  • Colony number 1 popped open in incubator, and could not be salvaged
  • Cast 1% TAE gel. Ran with 2 uL of NEB 1kb ladder.
Fig. 1 Colony PCR results, and gel purification of M2-15. The expected product size for colony PCR of M2-12(1C3) is approx. 1.6 kb. The expected size for M2-15mer PCR is 1630 bp.
  • Colony PCR indicates that colonies 2, 3 may be the correct size. However, the bands are slightly off shifted from each other. This indicates that one of the samples is probably wrong.
    • Will grow 5 mL cultures for colonies 1,2,3 to send for sequencing.
  • The M2-15mer PCR is inefficient compared to previous results. We may need to optimize, or just brute force it.
  • Excised the band corresponding to the M2-15 for gel purification.

Bacteria Culture

  • Set up 1x 100 mL culture each for M1-AB, BC, CA
  • Set up 5 mL cultures for colonies M2-12(1C3) colonies 1, 2, 3.