User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/06/29

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Ligation

ABSTRACT

  • Ligation of pBBRMCS5 plasmid and RFP both previously digested with ApaI and SacI.

  • Today Pablo and I run an electrophoresis gel to elucidate the proportions of DNA products that we are going to use for the ligation:

Lanes:

1. Ladder 10 Kb.
2. pBBRMCS-5.
3. pBBRMCS-5 digested by sacI.
4. pBBRMCS-5 .
5. pBBRMCS-5 digested by apaI and sacI.
6. Digested PCR.

Pae 29 jun 2011.JPG


  • So after analyze the gel, 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 did this reactions around 12:40 in the afternoon so we left them incubating at 20°C till 8 pm.

Transformation

ABSTRACT

  • Today, after we left incubating the ligation reactions, around 8 pm Pablo and I did the transformation of DH5α competent cells with the ligation reactions. According with the protocol of Transformación bacteriana that is in Pablo´s user page.


Four petri dishes were done:

 1)Transformed cells with reaction 1, more PCR product than vector.
 2)Transformed cells with reaction 2, less PCR product than vector.
 3)Transformed cells with reaction 3, control of ligation without PCR product.
 4)No transformed cells, control of transformation.