Single Worm PCR - faint bands after doing SW PCR (Aug/16/2006 )
I posted this in the Molecular section as well,
Hi All,
I work in a C.elegans lab and am trying to genotype some F3's from a cross I set up, using Single Worm PCR. I'm following the common practice of putting worms in lysis buffer (I used 3 worms in 3 ul of lysis buffer) freeze thawing, incubaing at temp for proteinase K, heat denaturing the proteinase K, then running PCR. I do get bands of the correct size, just very faint ones. I'm using primers given to me by my PI and the annealing temp she swears works for her. again I do get product, just ridiculously faint product after 30 cycles. Any suggestions?
Neil
I posted this in the Molecular section as well,
Hi All,
I work in a C.elegans lab and am trying to genotype some F3's from a cross I set up, using Single Worm PCR. I'm following the common practice of putting worms in lysis buffer (I used 3 worms in 3 ul of lysis buffer) freeze thawing, incubaing at temp for proteinase K, heat denaturing the proteinase K, then running PCR. I do get bands of the correct size, just very faint ones. I'm using primers given to me by my PI and the annealing temp she swears works for her. again I do get product, just ridiculously faint product after 30 cycles. Any suggestions?
Neil
Isn't it quite difficult to heat inactivate proteinase K?? And the EDTA and SDS (if included) in the lysis buffer also inhibits PCR, I would use P/Ch extraction or at least ethanol washes to get rid of the inhibitors.
Thank you. The lysis buffer used here did not contain EDTA or SDS. It was just Tris, KCl and MgCl + proteinase K (20mg/ml).
I denature proteinase K at 95 C for 15 minutes. I've heard that should be good enough. I too was under the suspicion that it woud take more I would Phenol/Chlorofom extract, but when you only use 3 worms as template you would never see the pellet.
I posted this in the Molecular section as well,
Hi All,
I work in a C.elegans lab and am trying to genotype some F3's from a cross I set up, using Single Worm PCR. I'm following the common practice of putting worms in lysis buffer (I used 3 worms in 3 ul of lysis buffer) freeze thawing, incubaing at temp for proteinase K, heat denaturing the proteinase K, then running PCR. I do get bands of the correct size, just very faint ones. I'm using primers given to me by my PI and the annealing temp she swears works for her. again I do get product, just ridiculously faint product after 30 cycles. Any suggestions?
Neil
Isn't it quite difficult to heat inactivate proteinase K?? And the EDTA and SDS (if included) in the lysis buffer also inhibits PCR, I would use P/Ch extraction or at least ethanol washes to get rid of the inhibitors.
I'm not familar with nematodes. I found this in www: http://www.wormbook.org/chapters/www_SNPsi...wopointmap.html
Their protocol looks good, if you're not already use it, so I would increase at least lysis buffer amount. If you see no pellet it does not matter, important is if the PCR or whatever is working afterwards. I work with insects (insect eggs and recently with thrips, both rel. tiny stuff) there is rarly a pellet, but DNA is there.