PCR Rescue: Making One Band From Many
It’s the molecular biologist’s version of ‘I have good news. . and bad news’.
The good news is that I amplified the DNA band of interest. The bad news is that I amplified these other bands as well! Oh, and this smear. What to do?
Typically you might try and cut out the band of interest and gel-purify – an issue if it’s a lower intensity product and most of the DNA is lost in the subsequent purification. Or the products are more closely spaced than your dexterity allows.
An alternative I’ve used for many years is band-stab PCR. This is an excellent technique to specifically re-amplify the DNA of interest and increase yield. With only one product generated in the subsequent PCR, the remaining PCR reaction volume can be rapidly cleaned up without gel purification.
How to do it?
1. When I notice multiple bands, I put my gel aside and go and set up a new reaction (I make up a 50µl reaction if I want a lot of this fragment). I add water in place of the usual DNA volume.
2. I then go back and place my gel on the transilluminator and gently blot off moisture with Whatman 3MM paper or Kimwipes.
3. Using either lower power or longer wavelength U.V. to avoid DNA damage, I then take a 20G syringe needle and, visualizing the DNA bands, stab the desired band of interest 2-3 times. No need to pick up any gel – just stab the band as you see it.
4. Next, I swirl the needle in my new reaction for a few seconds, then cap the tube.
5. Then I run the PCR amplification again using the same reaction, but only 20 cycles this time.
That’s it! You should then get a nice clean band corresponsing to the one you stabbed with the needle.
One band from many and one of my favorite techniques. It’s no good for real-time PCR of course, but great for things like ITS primers , which may cross react with plant species.
If you try this, let us know how it works. Do you have any other techniques for rescuing a poor PCR reaction?
Reference
Bjourson AJ, Cooper JE. (1992). Band-stab PCR: a simple technique for the purification of individual PCR products. Nucleic Acids Res. 20(17):4675.
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John Mackay
John is interested in using molecular diagnostics to solve industry issues - working on samples as diverse as wine, truffles, grapevine and human. He also consults on PCR and real-time PCR assay development as technical director for the company dnature.
Nice!
I guess I’m going to try it out.
You’re not concerned that your DNA in the gel is already damaged by the first visualization using high power settings? Or do you run a second gel?
What is the reason to run only 20 cycles in the second PCR?
Thanks!
Marc
A nice tip…
I’ll sure try it one day if I need it.
greetings from Portugal
I think it depends on your gel doc system. If the UV light stays on for a while while you fiddle around with sizing etc., then yes it might get nuked. Otherwise 5-10 seconds under high power has been fine to go back and re-amplify. Running the band-stab reaction, I have usually run the first reaction alongside to confirm the band of interest has amplified.
20 cycles is to avoid overamplification (and thus getting more non-specific bands). The needle into a band (however faint) is going to be picking up thousands of highly specific template copies.
One final thing, I have occasionally stabbed around the gel area where the band *should* have been. . .and subsequently got the product of interest.
Hi John,
Nice… such a simple and sensible solution to faint band problems. Very clever.
After having wasted many hours this summer trying to get a single band from a PCR reaction with many products (and of course a bit of lovely smear) I stumbled upon this today. And I tried it out immediately. And it seems to work! I will do a better attempt tomorrow, and if it continues to work I will experience the happiest moment of my PhD so far.
Thanks for a great page!
Good suggestion. In addition, when doing gel purification, ethidium bromide coupled with UV backlighting reduces cloning efficiency ~90% in a minute, and degrades the amplicon as well. If you use SYBR Safe stain and the Safe Imager (470 nm) from Molecular Probes/Invitrogen, cloning efficiency and amplicon size is not affected. Plus SYBR Safe is non-toxic and non-mutagenic, so it can be rinsed down the sink in most water districts.
Wow, interesting and innovative method!
Wonder what’s the success rate like…
I’ve tried this with a couple of pcr products and it worked great. I’m adding 1 mM Guanosine to the gel (as suggested in an earlier article) to prevent damage, but I doubt it’s necessary. I’m considering doing this on all cloning PCRs as possibly a better way to reduce/remove the threat of background caused by the PCR template (compared to a DpnI digest or full gel extraction).
When I last ran into this issue, I cut my band out of the gel and used the freeze ‘n’ squeeze method to purify the DNA out. It was very quick and easy, and I got a very pure strong product after re-amp. I wonder if I would have gotten the same result with the stab method.
I think one thing to watch out for is, depending on amplicon size, to use a high fidelity polymerase in the first round because any errors will be hugely amplified in the second round, possibly confusing your sequencing. It will probably not be an issue in most cases since you wouldn’t be resorting to this that often.
Tried it yesterday, combined with touch-down PCR and fewer cycles than I’d normally do – this technique works beautifully! The harvesting of template from the gel is so fast and simple that the risk of UV damage will be very minimal. Many thanks for the post!
One very happy molecular entomologist.