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Problem with Colony PCR


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3 replies to this topic

#1 saad

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Posted 25 February 2009 - 11:05 PM

Hi all

I did a colony PCR to check the plasmid and insert then on the agarose gel some samples appeared as many bands and I don't know what is the problem with the experiment.
Thank you in advance
Saad

#2 hanming86

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Posted 25 February 2009 - 11:58 PM

could be non-specific priming or too much DNA template.
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#3 noelmathur

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Posted 26 February 2009 - 06:11 AM

The important part is, if you got the band you expected. If you did then prep the plasmid and perform restriction digestion on the plasmid with two different sets of restriction enzymes to make sure that your insert is in there (and in right orientation, if its TA type of cloning)

#4 saad

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Posted 26 February 2009 - 09:21 PM

Thank you so much for your response
I sent the colony PRC product for sequencing - the forward primers worked but the reverse primers didn't work for all experiments- but when I look in database I found the insert match what I look for in database.
What I did is: I pick 4 colony from the same plate but the result is very different two of them got a good bands for insert and plasmid but the others appeared as many bands( look like a ladder), I don't know what is the problem? Is it in primers or

Cheers




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