User:Eduardo Vladimir Munoz/Notebook/UNAM Genomics Mexico 2011/2011/09/22

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High Stabilitiy Plasmid Development

Abstract

  • PCR for repC ligation corroboration

PCR

Strains 2, 10 and 11 presented restriction bands different to the rest of the strains (which were false positives). PCR will be performed with the repC speciffic primers Upper_repC (5207) and Low_repC (5208) to corroborate the insertion of repC to pSB1AK3.


Reaction + Control - Control
DNA PUX19XKR5 B0015 in pSB1AK3 Strain 2 Strain 10 Strain 11
PCR Reaction Volume
H2O mQ 33μL
10x PCR buffer Taq Pol. 5μL
8mM dNTP mix 5μL
Upper_repC 5μM 2μL
Low_repC 5μM 2μL
Sample DNA 1μL
Platinum Taq DNApol 0.5μL
Total 50μL


PCR proceded as following:

  1. Enzyme activation and Denaturation 1min at 94°C
  2. Amplification 30 cycles
    1. Denature 94°C, 30s
    2. Anneal 55°C, 30s
    3. Extend 72°C, 1min 50s
  3. Hold 72°C, 5min
  4. Storage 4°C, ∞