User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/09/15

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Parts Assembly-PCRs

ABSTRACT

  • PCR of PFOR2, HydEF1, HydEF2 and HydG

  • Today Fabricio and I went to ask for advice to do our PCRs, the problem was that many reactions were successful at the first time but when we want to reproduce them they were unsuccessful. So Isabel (one investigator at Genomic Ecology lab) directed us in some aspects of the process. She recommended us to lower denaturation temperature down to 95 °C and increase the time of it to 5 minutes so the plasmid can be open enough to let the primers anneal. She also recommended us use DNA extracted directly from bacteria, and dilute it in a proportion of 1:100.
  • So taking this into account I did 6 PCR reactions to amplify HydEF1 and HydEF2 regions with the following proportions:
H2O 24.5 μL
Buffer HF 10μL
dNTPs 10 mM 1 μL each
Primer Fwd 5μL
Primer Rev 5μL
Template DNA 1μL
Phusion Pol 0.5μL
Total 50μL

I made 3 reactions to amplify HydEF1 and another 3 to amplify HydEF2.

Cycles Gel Lanes Gel
  • 95°C 5min
  • 98°C 10s 30Cy
  • 55°C-60°C-62°C 30S 30Cy
  • 72°C 2 min 30Cy
  • 72°C 10min
  • 4°C
  • Lane1 Ladder
  • Lane2 HydEF2 55°F
  • Lane3 HydEF2 60°C
  • Lane4 HydEF2 62°C
  • Lane5 HydEF1 55°C
  • Lane6 HydEF1 60°C
  • Lane7 HydEF1 62°C
Gel15sept.jpg