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Re-PCR problem


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#1 EcoliO157

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Posted 11 May 2009 - 10:16 AM

Hi,

I have PCR products of various sizes with the target size being 1.1kb.
So, I gel extracted that fragment(fairly faint in intensity) and then re-ran the PCR. A lot of non-specific products were gone, but i still had a doublet, one at 1.1kb and ~800 to 900 bp right below.
Anyone has an explanation? You wouldn't direct sequnce these right?
If the two bands are so close together, is it possible to cut one band(meaning pure, homogeneous template) without carrying over the other band?

My guess is that there is another primer binding site internal to the intented binding site. causing a doublet to show up. Or during cutting of gel, a carry-over of some sort was enough to be detected by PCR.

#2 BGS

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Posted 12 May 2009 - 03:57 AM

Hi!
When I get any unspecific products in my PCR samples, I cut the wanted band out of the gel, purify it with a commercial kit and send it to sequencing. It works fine for me. I would not send samples where you can see more than one band to sequencing.
As to what is the cause of more than one band, there could be many explanations.
Hope this is of any help. Cheers

#3 EcoliO157

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Posted 12 May 2009 - 08:13 AM

thanks for the suggestion.

However, my problem is that the bands are to close together to cut out from the gel and also, the target band is not very bright, so it needs 2nd PCR to get more product for direct sequencing.

Another question i wanna add is that my PI is always against direct sequencing whereas I do direct sequencing and then cloning if i need to. I think it may be just a personal preference.


Hi!
When I get any unspecific products in my PCR samples, I cut the wanted band out of the gel, purify it with a commercial kit and send it to sequencing. It works fine for me. I would not send samples where you can see more than one band to sequencing.
As to what is the cause of more than one band, there could be many explanations.
Hope this is of any help. Cheers



#4 microgirl

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Posted 12 May 2009 - 09:39 AM

Can you do a nested PCR on your first PCR sample?

Hi,

I have PCR products of various sizes with the target size being 1.1kb.
So, I gel extracted that fragment(fairly faint in intensity) and then re-ran the PCR. A lot of non-specific products were gone, but i still had a doublet, one at 1.1kb and ~800 to 900 bp right below.
Anyone has an explanation? You wouldn't direct sequnce these right?
If the two bands are so close together, is it possible to cut one band(meaning pure, homogeneous template) without carrying over the other band?

My guess is that there is another primer binding site internal to the intented binding site. causing a doublet to show up. Or during cutting of gel, a carry-over of some sort was enough to be detected by PCR.






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