Unnatural smear in PCR product - (Jul/17/2007 )
Hi,
I have an interesting problem. I'm trying to amplify a 2kb region without any mutations if possible. So I'm trying Pfu nowadays. The expression level of my target transcript is too low, so I first do a PCR from the cDNA, then isolate the single but faint band. Finally, I use that product as a template and do the reaction again.
The problem is, I have a smear starting just under the wells and extending until 50 bp marker line. Can you think of any explanation for that?
Thanks in advance
How many ul did you load inside the well?
Hmm.. very unclean PCR.
sounds to me like endonucleasic activity.
Hmm.. very unclean PCR.
I loaded all the product, about 25 ul.
sounds to me like endonucleasic activity.Thanks for the idea but this only explains the smear below 2 kb, doesn't it? I also have a smear up until the wells, which is much longer than the 10 kb marker band.
Hmm.. very unclean PCR.
I loaded all the product, about 25 ul.
sounds to me like endonucleasic activity.Thanks for the idea but this only explains the smear below 2 kb, doesn't it? I also have a smear up until the wells, which is much longer than the 10 kb marker band.
Seems logically!! But what about DNA contaminations, that are endonucleasic digested. I suppose that u performed a negative control that was actually negative.
Another idea is the high amount of PCR-product used for electrophoresis. Why so much? As far as I know, normally 5µL are used after 25-35cycles.
And the last idea is protein contamination. In plasmid mini preps you often got these background smear over the whole lane, resulting from incomplete protein clean-up.
That are all my ideas - until now.
how faint is this band?
Can you use it in the ligation? Run 8 or more tubes of the primary PCR amplificatioin, pool the lot together and concentrate down the DNA. It should be enough for a ligation.
As for your reamplification problem, did you gel purify your single band? Any secondary PCR product, even if invisible on gel will work as a template and can cause problems.
Finally, 25ul of the primary amplification is far too much to use as a template for the secondary amplification
I have similar experience by using pfx, when I turn to normal Taq, every thing is OK
Can you use it in the ligation? Run 8 or more tubes of the primary PCR amplificatioin, pool the lot together and concentrate down the DNA. It should be enough for a ligation.
As for your reamplification problem, did you gel purify your single band? Any secondary PCR product, even if invisible on gel will work as a template and can cause problems.
Finally, 25ul of the primary amplification is far too much to use as a template for the secondary amplification
The product was gel purified but I didn't use all the primary product for the secondary PCR.
This may be the problem then, but I cannot use Taq since it causes too many point mutations. I think I'll try ExTaq instead.