problem with PCR --using fragment as template - (Apr/08/2005 )
QUOTE (littlecell @ Apr 9 2005, 04:03 AM)
hi, ananda,
i had the same problem two months ago---amplify my band from gel extraction with the same primers, but got only smears.
i tried everything but all failed. at last i found there's something wrong with the UV light used to cut the desired band. the UV is too potent and the DNA band was so seriously damaged that it could not be used as the template to amplify again with the same primers.
so my suggestion is : expose your gel under UV as short as possible. if there is only one band ( 500 bp) in your pcr product, don't use gel extraction!!! just use pcr purification kit to get 500 bp. to do this, you could run only a little fraction of your pcr reaction product on the gel to see if there is only one band. then use the clean 500 bp product to reamplify with the same primers.
good luck!
i had the same problem two months ago---amplify my band from gel extraction with the same primers, but got only smears.
i tried everything but all failed. at last i found there's something wrong with the UV light used to cut the desired band. the UV is too potent and the DNA band was so seriously damaged that it could not be used as the template to amplify again with the same primers.
so my suggestion is : expose your gel under UV as short as possible. if there is only one band ( 500 bp) in your pcr product, don't use gel extraction!!! just use pcr purification kit to get 500 bp. to do this, you could run only a little fraction of your pcr reaction product on the gel to see if there is only one band. then use the clean 500 bp product to reamplify with the same primers.
good luck!
thx! your explanation sounds very reasonable! are there any insights into the UV destruction of DNA?
infact i have used the pcr product(500bp) as template but without purification. it works but the band is weak.
-ananda-
QUOTE (wally @ Apr 9 2005, 07:22 PM)
I'm interested in the last lines of your post, because it also happened to me: how it can be that the smear is larger than the template???
Cheers
W.
Cheers
W.
maybe that's a black-box question.
Should the template form a template dimer? or even cross over each other
-ananda-
QUOTE (ananda @ Apr 10 2005, 08:32 AM)
QUOTE (wally @ Apr 9 2005, 07:22 PM)
I'm interested in the last lines of your post, because it also happened to me: how it can be that the smear is larger than the template???
Cheers
W.
Cheers
W.
maybe that's a black-box question.
Should the template form a template dimer? or even cross over each other
Yes, in fact, in another thread on the same forum, they say that when an internal repeat is in the template (as in my case), then the template could act as a primer in itself! And this could also explain why I find sometimes chimeric sequences!
thank you all!
W.
-wally-