Team:UCLA/Notebook/Spider Silk Genetics/21 August 2015

iGEM UCLA




8/21/2015

Gel Purification for M2 monomers

  • Used Zymo Kit. Concentrations around 25 ng/uL.

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

  • Used same ICA protocol as previous.
  • Initiator incubation for 45 minutes.
  • 3 minute ligation per monomer.
  • 100 uL washes.
  • Elute in 15 uL of 0.01% Tween-20 at 95 C for 5 min.

PCR amplification for M1-12, M1/2[1:1]-12, M1/2[2:1]-12

  • 2x 50 uL reactions for each.
  • For M1-12, used a 1:100 dilution of previous PCR product, for a total of ~100 pg added to reaction.
  • For M1/2[1:1]-12 and M1/2[2:1]-12, used 1 uL of direct ICA eluate.
M1-12 M1/2[1:1]-12 M1/2[2:1]-12
5x Q5 Buffer 10 uL
10 mM dNTPs 1 uL
10 uM For (F-03) 2.5 uL
10 uM Rev (G-03) 2.5 uL
Template 2 uL (~100 pg) 1 uL 1 uL
ddH2O 31.5 uL 32.5 uL 32.5 uL
Q5 Polymerase 0.5 uL
Total 50 uL
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

  • Ran PCR products on a 1% TAE gel, used 2 uL of 1 kb ladder.
Fig 1. PCR amplification of different 12 mers. The expected product is 1324 bp for all reactions.
  • Excised the bands from M1/2[1:1]-12 and M1/2[2:1]-12 for purification.
  • The expected product in M1-12 is present, but in too little of a quantity to purify. May need to do PCR using a gel stab as template.