Team:UCLA/Notebook/Spider Silk Genetics/28 August 2015

iGEM UCLA




8/28/2015

Gel Purification for M1 monomers

  • Used zymo kit, eluted in 12 uL of solution.
  • Yields were typical, around 25 ng/uL

Sequencing for M1, M2 monomers

  • Due to the presence of single bp mutations in the previous sequencing results, we wanted to check the sequence of the monomers to ensure that they are correct.
  • Sent each monomer sample for sequencing.

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

  • 3x 50 uL reactions for each. Used 1 uL of the ICA eluate as template.
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 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
  • Visualized on 1% TAE gel with 2 uL of 1 kb ladder.
Fig. 1 PCR amplification of M1-12 and M1/2[1:2]-12. The expected size for both is 1324 bp. The bands corresponding to M1-12 are at a lower intensity that those of M1/2[1:2]-12. This is probably due to age of sample.
  • Excised the 1324 bp bands for extraction.