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purification of PCR product for cloning in a vector


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#1 sanku

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Posted 07 February 2011 - 07:28 PM

Hi, I am doing PCR cloning. When I am amplifying my insert. I am getting primer dimer too. I can purify by gel but because of low yield I don't want gel extraction. if I purify it using pCR cleaning kit, I know that I am not going to get rid of primer dimer. Can primer dimer affect restriction digestion and ligation. some times my 260:230 ratio of gel extracted PCR product and digestive product is also not good. I wonder how much these factors can have an effect on my cloning.
yours suggestions would be highly appreciable.
thanks

#2 perneseblue

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Posted 07 February 2011 - 08:34 PM

In theory primer dimers will affect the ligation reaction. The primers dimers will have the same restrictions site at their ends and some molecules will be able to ligate to the vector.

In practice, you can screen through several colonies to find your desired plasmid.

It would be better if you can gel purify. But if you can't, then you will have to screen through more colonies than usual.

Might it be possible to PCR amplify several tubes of your DNA fragment. Thus you start with more material, which should allow you to go through gel purification
May your PCR products be long, your protocols short and your boss on holiday

#3 Jordan W

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Posted 07 February 2011 - 11:19 PM

Hi, I am doing PCR cloning. When I am amplifying my insert. I am getting primer dimer too. I can purify by gel but because of low yield I don't want gel extraction. if I purify it using pCR cleaning kit, I know that I am not going to get rid of primer dimer. Can primer dimer affect restriction digestion and ligation. some times my 260:230 ratio of gel extracted PCR product and digestive product is also not good. I wonder how much these factors can have an effect on my cloning.
yours suggestions would be highly appreciable.
thanks


If you are planning on doing this a lot as we do, there is no better machine out there than the E-Gel clone well system.

http://www.invitroge...Extraction.html

Basically, you run your DNA down to another well, and physically suck out the correct band when it get to the downstream well. The yields are huge and can be easily digested. May I ask why you just simply don't TA clone your PCR product first then digest PGEM to get the right insert?

Jordan

#4 sanku

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Posted 08 February 2011 - 07:51 PM

Thanks to all.




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