Colony PCR - (Jul/23/2009 )
umam on Jul 28 2009, 04:33 PM said:
I have another question, how do I understand the orientation of my insert when I use insert specific primer with Sp6/T7?
Thanks again,
-Uma
I've tried colony PCR a number of times, and had it work only once. I now just do a culture and a plasmid prep. I have a feeling I was using too much colony, but I'm not sure.
umam on Jul 28 2009, 01:33 PM said:
Thanks again,
-Uma
You can't. You either need to sequence your PCR product, or do a colony PCR using Sp6 and gene-specific reverse primer. If you get a PCR product, you'll know the orientation of your insert.
Cheers,
Minna
Minna on Jul 28 2009, 10:22 PM said:
umam on Jul 28 2009, 01:33 PM said:
Thanks again,
-Uma
You can't. You either need to sequence your PCR product, or do a colony PCR using Sp6 and gene-specific reverse primer. If you get a PCR product, you'll know the orientation of your insert.
Cheers,
Minna
Thats what, if I use Sp6 and gene-specific reverse primer, how do I understand the orientation?