User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/09/05

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Parts assembly-PCRs

ABSTRACT

  • PCRs from of the sequences of PFOR2, HydEF1, HydEF2 and HydG.

  • Today again I did 4 PCR reactions to amplify our parts. I try to repeat the conditions where each PCR was successful. So for PFOR2 and HydG the reactions were at the following proportions:
H2O 23μL
Phusion HF Buffer 10 μL
dATP 1 μL
dGTP 1 μL
dCTP 1 μL
dTTP 1 μL
Primer fwd 5 μL
Primer rev 5 μL
DNA template 1 μL
DMSO 1.5 μL
Phusion HF pol 0.5 μL

I made two reactions to amplify PFROR2 sequence and another two to amplify HydG sequence.

The temperatures and times of the cycles were:

Initial denaturation: 98°C for 30s

  • 30 cycles of:
 -Denaturation: 98°C for 10s
 -Annealing: 60°C for 30s
 -Extension: 72°C for 1 min 

Final extension: 72°C for 5 min Hold 4°C


  • The proportions of the reactions to amplify HydEF1 and HydEF2 were:
H2O 24.5μL
Phusion HF Buffer 10 μL
dATP 1 μL
dGTP 1 μL
dCTP 1 μL
dTTP 1 μL
Primer fwd 5 μL
Primer rev 5 μL
DNA template 1 μL
Phusion HF pol 0.5 μL

The temperatures and times of the cycles were:

Initial denaturation: 98°C for 30s

  • 30 cycles of:
 -Denaturation: 98°C for 10s
 -Annealing: A gradient of temperature from 55°C to 62°C
 -Extension: 72°C for 1 min 

Final extension: 72°C for 7 min Hold 4°C

  • Helena ran the electrophoresis gel

PCR gel was run at 120 Volts for 60 minutes 1μL of dye was used 2μL of PCR product were loaded 2μL of ladder were loaded

1 Ladder Fermentas
2 HG1
3 HG2
4 HF1
5 HF2
6 PF1
7 PF2
8 HydEF1
9 HydEF2
10 Ladder fermentas