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making one band from many
This article was originally published at BitesizeBio here
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 on the next gel corresponsing to the one you stabbed with the needle (I run some of the previous reaction alongside).
One band from many and one of my favorite techniques. Its not going to help your real-time PCR :-) 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?
Bjourson AJ, Cooper JE. (1992). Band-stab PCR: a simple technique for the purification of individual PCR products. Nucleic Acids Res. 20(17):4675.