User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/06/28

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Digestion of pBBRMCS5


  • Once the product of the plasmid extraction is clean after we use the Roche Kit, Pablo performed a digestion test with only one of the enzymes ApaI, this digestion test goes well. This indicates that the new strain used is DCM- and ApaI is capable tu cut because the DNA is not methylated. The next step was to do the double digestion with both SacI and ApaI, so today Pablo and I did this digestion and another digestion test with only SacI. The following proportions were used:

Digestion with only SacI

H2O 2μL
Buffer 4 10x 1 μL
BSA 1 μL
DNA 5 μL
SacI 1 μL

Double digestion with SacI and ApaI

Buffer 4 10x 2 μL
BSA 2 μL
DNA 13 μL
ApaI 1.5 μL
SacI 1.5 μL

Both reactions were left incubating at 25°C overnight, tomorrow we will run an electrophoresis gel with this digestions and with the PCR product digested (corresponding to a fragment like this: preffix-RFP-suffix) to find out the proportions that we are going to use to do the ligation.

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