IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/06/29

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Digestion's gel

The digestions made yestarday were run in an electrophoresis gel(120 V * 35 min) to see the resuslts. The digested PCR from pSB1T3 also was run in order to see the proportions of the ligation, this PCR contains apaI-preffix-RFP-suffix-sacI.

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.

From this gel we concluded:

1. sacI is working well, which is seen in lane 2.

2. At least one enzyme is worfing in the double digestion, and we hope both of them

are working because we previously probed the functionality of apaI.

3. The are multiple PCR, but the rigth one is the most concentrated.

4. The are more PCR concetration than double digestion concetration.

Ligation

Paulina and I made the ligation of:

  • apaI-preffix-RFP-suffix-sacI, from pSB1T3(lane 5 of the previous gel).
  • pBBRMCS-5 backbone. (lane 5 of the previous gel)

According to the concetration of each one(which is seen in the gel) we decided to test to proportions of the ligation:

Reaction 1

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


Reaction 2:

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


Control

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

Transformation

At nigth we did the transformation expecting to see red colonies tomorrow. There were 4 transformation corresponding to the 3 previous ligations and one transformation with no DNA(contro). In the platting we also did a control: nothing to cultivate.

The transformation was made according to this protocol Tomorrow we'll if some colines grew



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