User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/06/30

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PCR

ABSTRACT

  • PCR of the preffix-RFP-suffix fragment os pSB1T3 plasmid.

  • Today we didn´t observe colonies in the petri dishes of transformed cells with the different ligation reactions, we think this is due to a bad PCR product that is not reacting well with the digested plasmid, and the ligation can´t occur. We had 3 tubes with 3 different extractions of pSB1T3 plasmid so for elucidate which was the best I first run an electrophoresis gel:

According to this gel we decide to use the tube corresponding to the third lane. Pablo and I did 3 PCR reactions with the following proportions:

PCR reaction, we did 2 of this reactions.

H2O 36.5 μL
Buffer Taq 10x 5 μL
Primer forward 2.5 μL
Primer reverse 2.5 μL
10 mM dNTPs 1 μL
DNA 2 μL
Taq 0.5 μL
TOTAL 50 μL


PCR negative control

H2O 38.5
Buffer Taq 10x 5 μL
Primer forward 2.5 μL
Primer reverse 2.5 μL
10 mM dNTPs 1 μL
Taq 0.5 μL

Tomorrow we are going to run an electrophoresis gel to check if the PCR reaction goes well.



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