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cloning of GroEL - (Jun/13/2010 )

hi, i have trouble with cloning a 1.65 kb gene, GroEL, into TOPO BLUNT vector (invitrogen).
i hav performed all the steps as stated in the protocol brochure given along with the kit.
but i have used DH5 alpha cells, that i hav prepared myself with 0.1 M CaCl2.
the selectable marker i use is amipicilin, and its conc is 100 mg/ml.
i get fewer colonies but upon performing restriction digestion with EcoR1 i get only my vector.
i've also performed colony pcr, still the same result, hence confirming the fact that i hav no insert.

the gene (GroEL) i amplify is from the endosymbiont wolbachia, of the filarial parasite W.bancrofti of B.malayi.
i guess, GroEL in this case is a single copy number gene and hence i pool 6-8 pcr pdts, gel purify them, use that as template and reamplify the gene in order to intensify my gene conc.

plz suggest wat i shd do......

-prathibha sivaprakasam-

is the wolbachia promoter included or do you just clone the CDS?



its juz the CDS alone.......

-prathibha sivaprakasam-

i actually am looking for genetic polymorphisms form a wide geographical distribution......
i hav tried direct sequencing stratigies, but it hasnt worked my way......
and also that my senior cum project-mate has already worked on 2 other genes (Ftsz & MIF) this way and has reported polymorphisms.....

-prathibha sivaprakasam-