IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/07/01

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PCR results

The results of the PCR made yesterday are shown in the following electrophoresis gel(120V x 35min).

Lanes:

1. Ladder 5Kb.
2. PCR 1.
3. PCR 2.
4. PCR 3.

Pae 1 jul 2011.JPG

As expected the two first reactions corresponds to the fragment of intersts and the third which is the control showing nothing.

Cleaning PCR

I mixed the two reactions into one and then I cleaned it with the High Purification PCR Products kit from Roche. The final volume was 50 microlitters and to see if nothing wrong happened I ran 3 microL in a Daniel's gel:

Lanes:

1. Ladder 1 Kb.
2. Daniel's stuff 1.
3. Daniel's stuff 2.
4. Daniel's stuff 3.
5. Clean PCR.

Pae 1 jul 2011 2.JPG

All Right!!

Digestion of the PCR

In order to repeat the procedure of the ligation by sticky ends of the PCR with pBBRMCS-5, I did again double digestion by apaI and sacI:

The ligation's proportions(microL):

H20 3
BSA 2
Buffer 4 10x 2
apaI 1.5
sacI 1.5
DNA 10
Total 20