IGEM:UNAM-Genomics-Mexico/2009/Notebook/iGEM 2011 UNAM-Genomics Mexico/2011/06/02

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Digestion Control
We ligated the product of the digestion to make sure the plasmid PBBRMCS-5 was indeed digested with both enzymes (XBA I and Hind III). Then we transformed the plasmid with E. coli DH5α.

We expect that the transformation will not yield many E. coli colonies (there is not a perfect digestion).

Ligation

 * H2O -> 5.5 μL
 * Buffer 10X with DNTPs -> 1 μL
 * DNA (Plasmid) -> 2.5 μL
 * T4 ligase -> 1 μL

Ligation Control

 * H2O -> 8 μL
 * Buffer 10X with DNTPs -> 1 μL
 * T4 ligase -> 1 μL

For two total reactions of 10 μL.

Leave the reaction for two hours at 25ºC

Transformation into E. coli DH5α
* Tube 1 -> Transformation Control (no Plasmid DNA added) * Tube 2 -> 4 μL of ligated plasmid DNA added * Tube 3 -> 4 μL of the ligation control added (no plasmid DNA added)
 * We have 3 tubes with 100 μL of competent E. coli DH5α cells:
 * 1) Leave in ice for 20 minutes
 * 2) Heat shock for 1 minute at 42ºC
 * 3) Leave in ice for 5 minutes
 * 4) Add 1 mL of liquid LB to each of the three tubes
 * 5) Incubate for one hour at 37ºC
 * 6) Plate in petri dishes with solid LB and gentamicin
 * 7) Leave overnight at 37ºC and check in the morning for colonies


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