I am planning to do a cloning with PCR product i.e., my insert for cloning. I have observed 50% loss of yield from gel extraction of my PCR product. I have no non-specifics in my PCR product. I was thinking if I can do a PCR cleanup rather than gel extraction to save my yields as I don't have non-specifics. But I am unable to get any information about any drawbacks from PCR cleanup? Can anyone suggest me how to go about this? Is PCR-cleanup better for specific PCR product or gel extraction better for better yields for cloning process? IS PCR cleanup more helpful provided if it does not hamper anything in the process of cloning? Kindly let me know.
Hi, If you dont have any non specific products, then you can do PCR clean up instead of gel extraction. Mind you you do loose some product during PCR clean up as well but its far less than the loss in gel extraction.
Go ahead with PCR cleanup, it wouldnt hinder in the process of cloning.
Kotwal Shifa Bushra
Run through a gel can remove the non-specific amplicons, which you might not see on the gel. Even through you lost 50%, you should still be able to clone it with the remaining DNA fragments. If you want to increase the yield, you can make 3 identical PCRs, and run on 3 lanes (on a gel) and then cut them all out and purify them.
Thanks Yuan. But my amplicons give no non-specific bands that is why I was looking for PCR clean up. My amplicons give me a thick single band. Even your idea may work for me. Anyway Thank you for responding.
Thanks Tanuka. Even I was thinking for the same. But needed little confirmation whether What I was thinking is right or may be I am wrong.
Hi,
I would like to let you know that PCR clean-up is an alternative to gel extraction (but depends when to actually use) and you are absolutely right that you will lose DNA conc. via gel extraction. Now, results depend/vary in each case, however, I would recommend you to give it a try. I hope this will work.
Hope this would be helpful.
Thank you.
Hello, you do not need to do gel extraction if you have a very strong single band on your gel. You will lose a lot of product during cleanup no matter what, but gel extraction is worst. What I do now is add more than 100 ul PCR reaction to a PCR cleanup kit and use 35-40 ul warm elution buffer (65C) to elute from the column. This usually gives me 30-50 ng/ul.
It depends. If you use Gibson assembly for cloning (or some other really efficient approach), then simple PCR purification would work. If you do ligation, then gel isolation is better. If your template is a plasmid encoding the same selection gene as the vector you are planning to use, then gel purification is essential.
@ Micheal, In the MACHEREY-NAGEL Kit (MN kit) that I use, it is given 15ul-30ul for elution. So, I use 20ul. Is it better if I elute in 30ul?
If your PCR product is pure, you do TA cloning, just Cleanup, then clone.
If you need enzyme digestion, cleanup PCR product firstly, then digest PCR product, purify the fragment by gel extraction, ligation, transformation.
If you do cloning with assembly kit, cleanup your sample, then clone.
What about using low melting agarose and dissolving it with phenolics and further precipitation with ethanol? Have you tried it?
I believe yield is good enough.
Hello,
If you don't have any non-specific band, don't waste your time with a gel extraction.
I use the same kit (Macherey-Nagel Nucleospin gel and PCR Clean-up). All your questions have an answer in the user manual (P.11, PCR clean-up is better than gel extraction but the results presented here are funny ... Because I always observed a 50% loss unlike manufacturer's observation ...). For elution, Michael Paulsmeyer give you a good advise, use 65C warm elution buffer. For my part, I prefer to repeat elution twice (2 x 15 ul of warm buffer, there are also some good advises P.12 and 13).
Good luck !! Sometimes cloning is a nightmare ^^
Hey Thanks,
Even I follow the same format, like pre-warm the elution buffer and keep it for 10min and elute it twice. It really worked for me. But loss was not 50%, it was hardly 10-15%. But, Thank you all whoever has answered and helped. This was really a very good time we communicated to each other for helping each other.
I’ve used gel extraction & column purification. Column purification should work well for you if you have one clean band. It is faster & we do feel that less product is lost. We add 5x buffer QG & 1x Isopropanol, spin, clean, dry & elute. I only elute in 35-40ul so it’s more concentrated. I’m running a gel now to check if my digestion is linear & clean enough to do column purification. We do use the products for cloning. Good luck!
Purification of DNA from a PCR reaction is typically necessary for downstream use, and facilitates the removal of enzymes, nucleotides, primers and buffer components. The desired DNA in the aqueous phase can then be separated from other contaminants by ethanol precipitation. ...
If you have a single PCR amplified band then you can use hydroxyappatite columns to purify DNA. These are available as commercial kits. In case of multiple bands, you have to excise the band of interest from the gel and purify it via gel extraction kits or procedures.
Use some good company like Qiagen or Thermo PCR purification kit. These will yield 90-95% yield. Use pre-warmed elution buffer at 50oC. Use at least 2 round of elution with 5 min resting time and max centrifugation allowed for 2-10 min.
There are even PCR cleanup kits (resin or column based). I like the ones from Qiagen. I do always run a small part of the reaction on a gel (just to make sure it really is one nice band) then use the rest of the sample for the PCR cleanup.
If you start with more DNA than you need, a small loss in yield should not be a problem.
One way to streamline your process would be to use a polymerase and enzymes from a company like New England Biolabs, where you can digest your PCR product still in the PCR buffer. That saves on a cleanup step.
Or, you can TOPO clone your PCR product directly.
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