User:Eduardo Vladimir Munoz/Notebook/UNAM Genomics Mexico 2011/2011/09/13

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High Stabilitiy Plasmid Development

Abstract

  • repC-PCR with B0015 ligation colonies replating.
  • repC-PCR with B0015 ligation plasmid extraction.
  • Ligation of P1004 with B0014


Replating

I replated four colonies (1-4) that grew after transformation with the ligation of repC-PCR with B0015 in pSB1AK3 in a 6:4 ratio, and ten colonies (5-14) from the ligation with a 7:4 ratio.


Plasmid extraction

I extracted plasmid using Miguel's protocol from colonies 1, 2, 5, 6 and 9. I will corroborate plasmid concentration tomorrow with an electrophoresis gel.


Ligation

Since the ratios 7:7 and 9:5 didn't work for the ligation reaction, the ratios 5:9 and 11:3 will be tried:

Reaction 5:9 11:3
P1004 5μL 11μL
B0014 9μL 3μL
T4 DNA ligase 0.5μL 0.5μL
5x ligase buffer 4μL 4μL
H2O mQ 1.5μL 1.5μL
Total 20μL 20μL



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