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

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