Team:Panama/28 September 2010

September 28

Digestion of Rhlab with Pst1

Since the Rhlab gene is too big, we haven´t been able to complete the sequencing necessary for us to know if we're on the right track. We think that we can test it with a PstI digestion. We have checked the expected cuts with the NEBcutter (of NEB)and it is possible to but the Rh1ab gene with Pst1. After this we have to do:

-Electrophoresis

Clone Rh1ab into the plasmid.

-This week

PCR to show the RH1ab gene.

Mutagenesis (Next week)

The gene was purified with the Roche Kit: High Pure PCR Product Purification Kit

The purification of the gene RH1ab was done because it is a requirement in order to be able to use with the pGEM-T Easy Vector system and to clone it into a vector to later do the mutagenesis

Run a gel to see the disgestion of the gene and to verify the cut sites of Pst1

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1) We don't see the expected bands, possibly because we aren't using a purified PCR product

2) We can't see the product. We will have to repeat the ligation

3) We observe the band at the expected size

Measurement of the concentration of the purification: 96.1 ug/ml

Cloning Protocol of the pGEM-T Vector System

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