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Cloning of genes - (Apr/09/2008 )

If we have a cDNA preprared from the mRNA but we donot know what are the restricition sites are present in it and how it can be cloned into the vector for sequence analysis, either to clone it into TA cloning vector.
or there are some others method to clone it.

second how to methylated the cDNA, does the PCR produce with we amplify is also methylated.



first of all: do you have the sequence of your gene of interest, or at least part?

If you have the sequence, the easiest way to clone it is to make a PCR using primers spanning the entire coding region of the gene and then TA clone it. The utility of this, however, depends on what you want to do next!

About methylation: I know you can do bissulfite treatment to DNA (at least genomic), which I thing will convert unmethylated cytosines into uracil, but not methylated ones, but I have no experience in it. I'm not sure that was your question!


As suggested by SLAR, you can amplify with a new set of primers containing the required restriction sites.