Team:UCLA/Notebook/Spider Silk Genetics/10 July 2015

iGEM UCLA




7/10/2015

BBa_K525998 Miniprep

  • Used Zymo kit to miniprep the BBa_K525998 samples from yesterday.
Concentration (ng/uL) A 260/280
1 282.12 1.80
2 276.66 1.83
3 351.73 1.83
  • Sent samples for sequencing.

ICA for 6-mer. 9-mer

  • Used 1 uL of 50 nM initiator.
  • Used 50 ng of MaSp2 in each ligation.
  • 50 uL washes.
  • used 1 uL of 50 nM terminator
  • Eluted in 15 uL 0.01% Tween-20.

PCR amplification of ICA 6-mer, 9-mer

  • Used 0.5 uL of each solution as template.
  • Amplified with post-elution primers.
Volume (uL)
5x Q5 Buffer 5 uL
5x GC Enhancer 5 uL
F-03 1.25
G-03 1.25
dNTPs 0.5 uL
Template 0.5 uL
Q5 Polymerase 0.25 uL
ddH2O 11.25 uL
Total 25 uL
98 C 30 sec
98 C 10 sec
66 C 15 sec
72 C 15 sec
repeat from step 2 28x
72 C 2 min
12 C hold

Results

  • Used 1% e-gel, with 0.5 uL of 50 bp ladder.
Fig. 1 E-gel image of ICA 6- and 9-mer from elution and from PCR with post-elution primers. The expected product for 6-mer is 706 bp. The expected product for 9-mer is 1018 bp.
  • The resolution of this gel is somewhat too low to accurately size bands. We will run on a higher percentage gel later.
  • Elution products show nothing, but there are products after PCR amplification.
  • The presence of multiple bands may indicate non-specific amplification, or improper ICA.
    • Plan on doing temperature testing, and more troubleshooting.
  • Extension time was a bit short for a 9-mer.