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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

-jahan-

How many ul did you load inside the well?
Hmm.. very unclean PCR.

-timjim-

dry.gif sounds to me like endonucleasic activity.

-mikroangie-

QUOTE (timjim @ Jul 17 2007, 05:01 PM)
How many ul did you load inside the well?
Hmm.. very unclean PCR.


I loaded all the product, about 25 ul.

QUOTE (mikroangie @ Jul 17 2007, 06:21 PM)
dry.gif 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.

-jahan-

QUOTE (jahan @ Jul 17 2007, 05:48 PM)
QUOTE (timjim @ Jul 17 2007, 05:01 PM)
How many ul did you load inside the well?
Hmm.. very unclean PCR.


I loaded all the product, about 25 ul.

QUOTE (mikroangie @ Jul 17 2007, 06:21 PM)
dry.gif 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. wink.gif
Another idea is the high amount of PCR-product used for electrophoresis. Why so much? As far as I know, normally 5┬ÁL 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.

-mikroangie-

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

-perneseblue-

I have similar experience by using pfx, when I turn to normal Taq, every thing is OK

-rye-

QUOTE (perneseblue @ Jul 17 2007, 11:48 PM)
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


The product was gel purified but I didn't use all the primary product for the secondary PCR.

QUOTE (rye @ Jul 18 2007, 03:20 AM)
I have similar experience by using pfx, when I turn to normal Taq, every thing is OK


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.

-jahan-