User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/07/04

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Ligation

ABSTRACT

  • Ligation of pBBRMCS5 plasmid and preffix-RFP-suffix fragment both previously digested with ApaI and SacI.

Pablo and I did 3 ligation reactions with the following proportions:


Reaction 1: It has more PCR product than vector.

H2O 3 μL
Buffer T4 10x 1 μL
Vector 2 μL
PCR product 3 μL
T4 DNA ligase 1 μL


Reaction 2: It has less PCR product than vector. We did this reaction because the PCR product was more concentrated than the vector so maybe with the previous reaction there is the possibility that the PCR products ligate with each other and not with the vector.

H2O 4.5 μL
Buffer T4 10x 1 μL
Vector 2 μL
PCR product 1.5 μL
T4 DNA ligase 1 μL


Reaction 3: This is a ligation control without PCR product.

H2O 6 μL
Buffer T4 10x 1 μL
Vector 2 μL
T4 DNA ligase 1 μL

We left this reactions incubating at 16°C overnight.