PCR--11kb fragment - (Jul/01/2010 )
Hi all,
My supervisor has asked me to amplify a 11kb fragment from C. elegans genomic DNA. The primers' Tm are 56.7 and 55.8 degrees. I used System 2 of the Roche Expand Long Template PCR system and followed the instruction for the reagent concentrations (500 microM for dNTPs, 300nM for primers, 5microL buffer that contains 2.75mM MgCl2, around 200ng of genomic DNA and 0.75microL enzyme) and thermal cycling. At an annealing temperature of 56 degree I was able to get a long smear. At 58 degree annealing temperature there was nothing visible on the EtBr stained gel. >< I tried decreasing the template, or primer, or both, but nothing's working yet.
Please offer any suggestion/hints that you may have.... Anything will help as I'm banging my head against the wall right now. Thanks!!!!
Are you talking about Roche's plymerase which claims it can amplify up to 20K? last time I used it and got smaer, but then I lowered my extension time and the smear disappeared. my smear was above the band.
sanguinara on Jul 1 2010, 03:24 PM said:
My supervisor has asked me to amplify a 11kb fragment from C. elegans genomic DNA. The primers' Tm are 56.7 and 55.8 degrees. I used System 2 of the Roche Expand Long Template PCR system and followed the instruction for the reagent concentrations (500 microM for dNTPs, 300nM for primers, 5microL buffer that contains 2.75mM MgCl2, around 200ng of genomic DNA and 0.75microL enzyme) and thermal cycling. At an annealing temperature of 56 degree I was able to get a long smear. At 58 degree annealing temperature there was nothing visible on the EtBr stained gel. >< I tried decreasing the template, or primer, or both, but nothing's working yet.
Please offer any suggestion/hints that you may have.... Anything will help as I'm banging my head against the wall right now. Thanks!!!!
Curtis on Jul 2 2010, 02:15 AM said:
Thanks for the info Curtis!
Yep that's the one. My smear was pretty big, presumably smaller than the size of the desired band. But there 's no band visible.
I am right now cloning a 14-kb segment of DNA which I PCR amplified from chromosomal DNA using Hi Fidelity Taq. No need for specialty PCR mixes...
HomeBrew on Jul 2 2010, 01:39 PM said:
I'll give it a try! Thank you!