Team:UCLA/Notebook/Spider Silk Genetics/7 September 2015

iGEM UCLA




9/7/2015

Colony PCR for M1-15(T7)

  • Picked 16 colonies, resuspended each colony in 50 uL LB.
  • Used 1 uL of resuspended colony as template in PCR using Taq Red Master Mix.
Volume (uL)
2x Taq Red Master Mix 6.25
10 uM For (VF) 0.625
10 uM Rev (VR) 0.625
Colony 1
ddH2O 4
Total 12.5
95 C 3 min
95 C 25 sec
56 C 30 sec
72 C 1.5 min
repeat from step 2 30x
72 C 5 min
12 C hold
  • Visualize on 1% TAE gel, with 2 uL of NEB 1 kb ladder.
Fig. 1 Colony PCR for M1-15(T7). The expected band size is approximately 1.9 kb. There are only 2 colonies that might possibly have the correct sequence. Colonies 6 and 12.
  • Inoculated 5 mL of LB + chlor with 25 uL of resuspended colony 6, 12 for sequencing tomorrow.