IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/07/26

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July 26, 2010

EGFP has stayed elusive to cloning with PCR so we tried it again. Also we ran out of MicF and had to clone that again. We ran two reactions:

  • RXN 1
    • 5.0 uL 10X pfu buffer
    • 0.4 uL pfu DNA polymerase
    • 0.4 uL primer forward
    • 0.4 uL primer reverse
    • 1.0 uL 4X dNTP
    • 10.0 uL template (e.coli genome 20 ng/uL)
    • 32.8 uL dH2O
  • RXN 2
    • 5.0 uL 10X pfu buffer
    • 0.4 uL primer forward
    • 0.4 uL primer reverse
    • 0.4 uL pfu DNA polymerase
    • 1.0 uL 4X dNTP
    • 1.0 uL template (pXG-10 475 ng/uL)
    • 41.8 uL dH2O

The PCR setting that was used:

  1. 95 C: 5 min.
  2. 95 C: 30 sec.
  3. 51 C: 30 sec.
  4. 72 C: 1.5 min.
  5. 72 C: 10 min.
  6. 4 C: hold

Numbers 2-4 were repeated 32 times.

I also inoculated 1 white colony from each of the 4 plates made on 7/22/10. Each was inoculated into 5 mL of LB Broth with added 5 uL of CAP resistance added to them. They were then put into a spinner in the 37 C room overnight.


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