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Colony PCR - (Jul/23/2009 )

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umam on Jul 28 2009, 04:33 PM said:

Also- colony PCR is supposed to be reliable, I know a lot of peple who do it regularly,why would you call it unreliable?

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.

-fishdoc-

umam on Jul 28 2009, 01:33 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


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-

Minna on Jul 28 2009, 10:22 PM said:

umam on Jul 28 2009, 01:33 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


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?

-umam-



Thats what, if I use Sp6 and gene-specific reverse primer, how do I understand the orientation?


So if you have Sp6-insert-T7 and you make a PCR with Sp6 and T7, you'll get a product in any case. If you make a PCR with Sp6 and gene-specific reverse primer, you will get a product only if your insert is in "forward" orientation. If it is reverse, then both primer are amplifying in the same direction, and you won't get a PCR product.
I hope I could make myself clear...
Cheers,
Minna

-Minna-

Does anyone knows by first hand experience if Colony PCR works with gram positive bacteria? I was told by my ex boss that it doesn't work. But now my new PI told me that it should work. However, I ran a gel after a colony pcr and got no bands at all. But I am not sure if it is because of the method or because of a false positive transformant.

-planktonica-

If colony PCR is not working it doesnt mean that u are not having the insert.....do a Miniprep and then restriction and confirm on on a gel.........for me the colony PCR didnt work but after miniprep and digestion with restriction enzyme i found on gel that i had it..............
I admit that colony PCR is a robust way to know about your insert presence......but it may fail most of the times for reasons that am not getting....while sometimes it works..........it was a strange experience so now i have abandoned it and go to miniprep directly.........

-Adnan-
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