Team:UCLA/Notebook/Spider Silk Genetics/30 July 2015

iGEM UCLA




7/30/2015

Transformation Results

  • Colonies Present on M2-15(1C3) and M2-12(T7) plates.
  • Plan to do pick 4 colonies and analyze via colony PCR.

ICA for M1-9

  • 45 minute initiator incubation time.
  • 100 uL washes.
  • 4 min ligation time at RT.
  • Prepared the reaction mixtures ahead of time.
  • Elute in 0.01% Tween-20
    • 95 C for 5 min.

PCR Amplification for M1-9

  • 1x 50 uL reaction.
  • Used ICA eluate as template, amplified using post-elution primers.
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 1
Q5 Polymerase 0.5
ddH2O 32.5
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

Colony PCR for M2-12(T7), M2-15(1C3)

  • Picked 4 colonies from each. Resuspended in 50 uL LB. Used 1 uL of resuspended colony as template.
  • used 2x Taq Red for colony PCR
  • Total 12.5 uL reaction for each colony.
Volume (uL)
2x Taq Red 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 min
repeat from step 2 30x
72 C 5 min
12 C hold

Results

  • Cast 1% TAE gel. Used 2 uL of NEB 1 kb ladder.
  • Visualized M1-9 and colony PCR results.
Fig. 1Results of Colony PCR for M2-12(T7) and M2-15(1C3), and PCR amplification of M1-9. The expected size for M1-9 is 1018 bp. The expected size for M2-12(T7) is 1.6 kb. The expected size for M2-15(1C3) is 1.9 kb.
  • The PCR amplification of M1-9 was successful. The band at 1018 kb was excised for gel purification.
  • Colonies 1, 2, 3, 4 for M2-12(T7) all appear to be correct. Will choose 3 for liquid culture and sequencing.
  • Colonies 1, 2 for M2-15(1C3) appear to be correct. Colonies 3, 4 appear incorrect. Will send colonies 1, 2 for sequencing, as well as one more picked colony from the plate.

Liquid Culture

  • Prepared 3x 5 uL cultures for M2-12(T7) and M2-15(1C3) each.
  • Inoculated with 25 uL of colony resuspended in 50 uL of LB.