User:Jose Fabricio Lopez/Notebook/Logbook/2011/09/04

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Goal

  • PCR for trouble shooting about PCRs for isothermal assembly.

Troubleshooting

  • PCR with Taq Platinum invitrogen and 57°C for annealing.

The objectives of those changes are:

  • Change times for elongation.
  • Change Temperature for anneling.
  • Excellent yield by PCR.

Gel

Completely unsuccessful

Image:PCRwithTaq Control.JPG

We are trying to know what is happening with PCRs.


PCR with different parameters

So far, we have amplified HydG and PFOR1, since now we are trying a new approach. For that we made a simply PCR, this is without GC buffer and DMSO. PCR is for HydEF1 and HydEF2 ( new diluted plasmid with 10ng/μL )

55°C 60°C
HydEF1 HydEF2
HydEF1 HydEF2
Reagents 55°C 60°C
Buffer HF Phusion 10μL 10μL
dNTPs (10mM each) 4μL 4μL
DNA diluited 10ng/μL 1μL 1μL
Phusion .5μL .5μL
OligoFwd 5μL 5μL
OligoRev 5μL 5μL
H2O 24.5μL 24.5μL

Gel



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