How can I purify a PCR product that is smaller than 70 bp? I typically use a Qiagen gel purification prep to purify PCR; however, the manual states that fragments of that size are removed rather than purified. So, how do I get my fragment out?
Hi, Joe. I clone short stuff with these kits pretty often. It doesn't get rid of them all that much. If you are worried, just try it and see what you are getting. Keep in mind that gel purification kits are good at losing all DNA, not just short pieces :)
The Qiagen Nucleotide Removal kit will retain fragments >40 bp, if I remember correctly.
Hi, Joe. I clone short stuff with these kits pretty often. It doesn't get rid of them all that much. If you are worried, just try it and see what you are getting. Keep in mind that gel purification kits are good at losing all DNA, not just short pieces :)
Hi, I don't know if this will work properly: https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314774443672/litdoc28951562AA_20110831111511.pdf
Cheers, Nadine
Abstractly, can you not collect the effluent that is expelled from the Qiagen process, and reprocess it by a second method?
Hi Joe,
As Simone and Ivan has suggested, I will go ahead and try your purification kit. I am sure your recovery won't be the best, but if you have a big enough sample it should not be a problem. I have had a similar issue in the past, and I quite sure I ended up buying a purification kit from Fermentas, especially designed for small products (> 40 bp). They also have special instruction for purifying and recovering small products, like adding more buffer, using a smaller volume of eluent (and warmed up at ~30 C degrees) and so on.
All the best,
You can cut your small fragment gel (~70bp) and dissolve it with the QG buffer that you have from Qiagen and subsequently perform ethanol precipitation (co-precipitate with tRNA or glycogen if you have them in your lab) instead of passing through the column.
I also know some people cut off the gel fragments and crush/squeeze them on parafilm. The resulting liquid/fluid from the agarose is then collected (make sure collect the liquid without the traces of agarose pieces) using a pipette for cloning.
Alternatively, you may want to purchase kit from qiagen:
http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/dna-cleanup/qiaquick-nucleotide-removal-kit
You can try ethanol precipitation. Just add 96% ethanol with 1% sodium acetate (or only ethanol) centrifuge for 20 min in 4deg. remove ethanol and add 70% ethanol to wash the the pellet and centrifuge for 20 min in 4deg. Remove ethanol and dissolve the product. Of course this will be only working if you have a lot of product.
The QIAquick gel extraction kit works very nice. To be sure that you will isolate the correct band I would run my sample in a 2% agarose gel and at least 50ul from a PCR rx so ensure that you will see a well-defined band. To concentrate your sample, make the elution step on 30-35ul.
Magdalena: Ethanol precipitation will not be effective if the original poster is trying to purify the PCR products away from the primers. The primers will precipitate as well as the PCR fragment. Also, washing the pellet with 70% ethanol is not necessary unless a lot of salt is precipitated.
Hey Everybody,
I'm struggling to purify 35 bp long PCR product. I have to clone that it to a plasmid as well by double digestion. I cant find a suitable kit or protocol for that. Could anyone recommend me or have previous experience about cloning fragments less than 35 bp.
I'll be highly obliged...
Thank you very much,
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