Team:UCLA/Notebook/Spider Silk Genetics/20 May 2015

iGEM UCLA




5/20/2015

1. VF/R Amplification of MaSp Plasmid

  • Based on yesterday's results with amplifying 2CA using the VF/R primer pair, we decided to scale up to obtain enough plasmid to perform digestion tests.
  • We used the following protocol, and set up 2x 50 uL for amplification of 2CA.
Amount (uL)
5x Q5 Buffer 10
10 mM dNTPs 1
10 uM For 2.5
10 uM Rev 2.5
2CA (219 pg/ul) 1
5x GC Buffer 10
Q5 Polymerase 0.5
ddH2O 22.5
Total 50
98 C 30 sec
98 C 10 sec
66 C 15 sec
72 C 15 sec
repeat from step 2 17x
72 C 2 min
12 C
  • 2CA was diluted from 219 ng/uL 1:1000 in ddH2O to 219 pg/uL.

2. Results

  • We visualized the results on 2% TAE agarose gel using 2 uL NEB 50 bp ladder.
    Fig. 1 MaSp2 2CA amplified using VF/R primers. The expected VF/R product is 443 bp. The expected P/E product is 170 bp.
  • The presence of a smear under 50 bp may indicate incomplete PCR and leftover primers. We will try 20 cycles in the future to see if better amplification can be achieved.
  • We excised the bands at ~450 bp for gel extraction tomorrow.