Team:UCLA/Notebook/Spider Silk Genetics/4 September 2015

iGEM UCLA




9/4/2015

Miniprep for M1-15(1C3)/(T7) and Tamura(1C3)

  • Used qiagen kit.
  • Elute in 30 uL.

Restriction Digest Test

  • Digest with X,P to see insert size.
  • Digest 1 ug of plasmid in a 50 uL reaction with 1 uL of each enzyme.
  • Digest at 37 C for 1 hr 15 min, heat kill at 80 C for 15 min.
  • Visualize on 1% TAE gel with 2 uL of 1 kb ladder.
Fig. 1 X,P digest of M1-15(1C3), M1-15(T7), and Tamura(1C3) samples prior to sequencing. The expected insert size for all three constructs are 1.6 kb. None of the M1-15 colonies have the right insert. Both Tamura(1C3) colonies have the correct sized insert.
  • M1-15 colonies are wrong; don't send for sequencing.
  • Send Tamura(1C3) for sequencing with VF, VR primers.
    • Also send old Tamura(1A3) for sequencing as well.

Colony PCR for M1-15(T7)

  • Since no colonies are left on M1-15(1C3), will focus on M1-15(T7).
  • Picked 8 colonies from M1-15(T7) plate, resuspended in 50 uL LB.
  • Colony PCR with 1 uL of resuspended colony as template.
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 colony PCR results on 1% TAE gel with 2 uL of 1 kb ladder.
Fig. 2 Colony PCR of M1-15(T7) using VF/R. The expected band size is 1.9 kb.
  • Colony PCR shows that the 8 colonies picked are incorrect. Will pick more.