User:Eduardo Vladimir Munoz/Notebook/UNAM Genomics Mexico 2011/2011/08/25

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High Stability Plasmid Development

Abstract

  • Inactivations of the double digestions of repC and B0015.
  • Corroboration gels.
  • Ligation of repC and pSB1T3

Inactivations

Double digestions (DD) were inactivated for twenty minutes at 65°C, as indicated by the enzyme manuals.


Corroboration gel

Both reactions and their controls were loaded on the same gel:

Lane Load
1. DD repC-PCR E,S
2. DD repC-PCR E,X
3. B0015
4. DD B0015 E,P
5. DD pSB1T3 E,P
6. Ladder 500bp

DigesRepC-Back DoubleTer-Back.JPG

Ligation

I prepared two ligation reactions, with plasmid-insert mass concentration ratios of 1:5 and 3:3.

Ratio 1:5 3:3
DD repC-PCR E,S 5μL 3μL
DD B0015 in pSB1AK3 E,X 1μL 3μL
Buffer 10x 1μL 1μL
T4 ligase 1μL 1μL
Agua 2μL 2μL

Fibo prepared reactions of his own as well.