I've read different protocols, starting for the QuikChange one, and there's no call for cleaning up after PCR. However, I did clean up as suggested by other researchers, so the polymerase doesn't interfere. So I digested with DpnI and transformed, but I'm not getting any colonies. I included a positive and negative control and the antibiotic and competent cells are working fine. Also, the concentration of my plasmid after cleaning it up decreases a lot. Could it be that the problem?
Hi,
did you use elution buffer containing EDTA? sometimes EDTA will block restriction enzymes activity. The best is to elute in water. Also, are you sure thar you are using a strain dam+, that indeed metylate the DNS?
You can increase plasmid concentration by reducing the elution volume, incubating your DNA in the filter and passing through serveral times.
Hope it helps you.
Hi Miguel,
Yes, the strain we are using is dam+. Also, our buffer doesn't have EDTA, and I'm eluting in 30 microliters instead of 50. But I will try this time following your recommendation about the filter.
Thank you so much.
The following may help you...
https://www.genscript.com/dna-transformation-troubleshooting-guide.html
https://international.neb.com/tools-and-resources/troubleshooting-guides/restriction-enzyme-troubleshooting-guide
In terms of cleanup and Dpn1, it depends on the polymerase you're using (more specifically on its buffer). If you are using Phusion (https://international.neb.com/products/m0530-phusion-high-fidelity-dna-polymerase) then you can add Dpn1 directly to the completed PCR mix (it doesn't hurt to add some extra Cutsmart buffer at the same time), then carry out your Dpn1 digest (to a 50 ul reaction mix add 1 µl Dpn1 and 5 µl Cutsmart and incubate at 37°C for 1 hr then 80°C for 20 min).
However, if for example you are using Q5 polymerase (https://international.neb.com/products/m0491-q5-high-fidelity-dna-polymerase#Product%20Information) then you will need to perform a cleanup step before the Dpn1, as Dpn1 is not active in the Q5 buffer. This may apply to other high fidelity polymerases too.
I generally use Phusion, perform the Dpn1 directly on the PCR reaction mix (as above), then transform directly. This assumes that you are transforming commercially competent cells via heat shock. Supercompetent cells are best (if not essential) for this type of job.
To me it doesn't sound like your Dpn1 is a problem though, as when that is the case you tend to get lots of background from undigested template. That would also be the case if your template was not made in a dam+ strain (as Miguel alluded). More likely you are either a) not making much product or b) losing your product during the cleanup.
If it is b) then the process (Phusion, direct Dpn1, no cleanup) I've mentioned above may sort it. If it is a) I would advise looking at your primer design, as you may be getting primer dimers rather than product (a common problem with SDMs). Also if you design your primers with an accentric back-to-back overlap (rather than as mirror image primers, which are often advised by kits etc) you can achieve exponential amplification which will give you more product.
If you're happy that the primers are making a product, and you have been using supercompetent cells, but you still see no transformants you could try to electroporate (using electrocompetent cells). This generally gives higher efficiency, so it would work with low product recovery levels. BUT be aware that you would need a cleanup step before it to remove salts (you'd elute in water).
As mentioned by Miguel, you can improve your cleanup steps by allowing a bit longer incubation time (over lunch works nicely!) of the eluent solution (here water) on the column. I would also advise warming the eluent solution on a heat block before you put it on the column.
Finally, something I've often considered but have never tried, is that following Dpn1 digestion, you perform a ligation. This would seal the nicked overlaps of the product and should help transformation efficiency a bit.
Hope something there helps, good luck.
Cleaning up PCR products has a cost of reducing on concentration. However, it is advisable to clean up anyway as this removes all the components in the PCR mix from the plasmid allowing you to have a clean product for restriction digestion. I would advise that you try to increase on the concentration of the PCR product before cleaning up so that you have enough template in the restriction digestion.
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