2 replies to this topic

[chrom]

Hi everyone,
I face a wierd problem. My gene about 630bps long gets amplified from cDNA library when I use Taq DNA polymerase, but i cant see any amplification when I use XT-5 polymerase from genei. I have used the same reagents and conditions. I need to clone this gene and hence want it to be error prone. I would highly appreciate ur suggestions. Thanks

[Unagi]

I'm not familiar with XT-5, but perhaps it needs to be run in a specific buffer (or at least a different one to the one you are using for the Taq reaction). Does the XT-5 have different properties which need to be taken into account during cycling? (eg: is it a hotstart enzyme and needs an initial incubation?)

chrom, on Sep 23 2009, 07:44 PM, said:

Hi everyone,
I face a wierd problem. My gene about 630bps long gets amplified from cDNA library when I use Taq DNA polymerase, but i cant see any amplification when I use XT-5 polymerase from genei. I have used the same reagents and conditions. I need to clone this gene and hence want it to be error prone. I would highly appreciate ur suggestions. Thanks

[chrom]

Hey thanks unagi for ur reply. The enzyme had trouble binding the template, so i did a hot start and it worked . But I dont really think it a hot start enzyme. I have already run many pcrs using this enzyme without hot start and it always worked.
But to conclude...i got my pcr product finally
thanks