amplification of GroEL from Wolbachia! - amplification of GroEL from Wolbachia! (Jun/28/2010 )

hi all!

am working identifying genetic polymorphisms in the endosymbioant Wolbachia of Brugia malayi and/or Wuchereria bancrofti.
the gene am trying to amplify is GroEL, the HSP 60 ptn, needed for the proper folding of proteins.

its a 1.65 kb gene for which i hav designed primers and have tried to amplify.

two months ago, i used to get a very faint band of the required size namely 1.65 kb, but however when am trying to amplify it now, am getting only smearing in the entire lane.

am using the same DNA which i had used then, however the DNA was stored in -30C, so i guess the possibility of degradation should be ruled out.

what else cud be the reason of smearing??? please help me out in this regard.......!!!!!!!

Actually, I do not think you can rule out DNA degradation. Freezing and thawing your genomic DNA will shear it into smaller pieces. If at all possible, I would recommend purifying the DNA again and re-runing the PCR.

Good luck.

thanks Ivan, but the scenario is that i had last used my DNA during last week of April and from then on it was left untouched......

that's the reason y i told u that the possibility of DNA degradation can be ruled out.....!!!!!!