Hi Everyone
My PCR on DNA extracted from insects isn’t working. I can see the band of DNA when checked for sample integrity. Primer bands are also visible. I have tried gradient temperature range but the DNA amplification isn’t visible. Any insights will be helpful.
@Ivan Delgado
You were a PCR Genius, Can you help her out ?
Hi Navneet,
Yours is a classic PCR troubleshooting case. Here is what you do:
You need to first and foremost make sure the PCR itself is working (i.e. reagents, primers, etc are all good). To do that you need to get control DNA that you know is good and run a control PCR on it. By control DNA I mean DNA that has been purified before and used to successfully run a PCR reaction. If you do not have that you may have to buy it. Companies sell control DNAs for this purpose. And you need a control PCR reaction. By this I mean a generic PCR reaction that has been around for years and is known to be robust and works under almost all conditions, even when the DNA is partially degraded. Such PCR reactions are known by other names like “internal control PCRs”.
Once you have control DNA, and a control PCR, you run the PCR. You would of course get a positive result. This positive result, likely measured by running the PCR reaction on an agarose gel, should show very strong bands on a gel. If you don’t then your problem is somewhere else, for example your PCR machine may be having issues. Or there is an issue with your technique (introducing contaminants that lead to PCR inhibition, etc). It happens.
Once you have run a successful control PCR with a strong PCR signal then you test the control PCR on your purified insect DNA. If the PCR fails then it is the DNA. Many PCRs fail when the DNA is not pure enough. Just some residual protein left over in purified DNA can inhibit PCR amplification. If this is the case you need to change your DNA purification protocol. You need to use a DNA purification protocol that yields 100% pure DNA. Kits that use columns to purify DNA are usually the best way to achieve this.
Once you get the control PCR to work on your purified DNA you run your own PCR on it. If it fails then you know it is your PCR that is the problem not the DNA. In that case you need to go back and re-design your PCR. One good trick to obtain a good and robust PCR reaction is to use one the PCR design programs that are free online and design at least two or three pairs of primers to test. More often than not at least one of the primer pairs you designed will work much better than the others.
This may sound like a lot of work but if you follow this steps you will likely save time in the long run troubleshooting a PCR reaction.
Thanks for the detailed reply Ivan & thanks Argerine for connecting. I’m already at step 3 because PCR is working with my Positive Control Sample. My sample was treated with Proteinase K & I checked the integrity of the sample with a pre run before PCR. The sample looked healthy and has been desalted too. What should the next step be ?
If you already have a PCR that works very well with your control DNA then the problem is your DNA.
Treating a sample with proteinase K is fine but may not be enough. Some protein may still be left over that could inhibit the PCR. My recommendation is to column purify your DNA to be 100% sure it is pure. You can buy a kit for that. It is a little more work/cost but it should make a big difference.
Isolating DNA again from a new/fresh sample is a good idea. If you have access to a spectrophotometer, like a Nanodrop, read the quality and quantity of your DNA. You want a 260/280 ratio of 1.8 to 2.0 and around 20 ng to 50 ng of DNA per PCR reaction.
If your PCR is working no need to play with that. Focus on getting a new better DNA.
Thanks Again Ivan
My sample’s 260/280 ratio was well in order. But I’ve proceeded with a fresh sample today. Used desalting columns this time and hoping to get this work right way this time.
Ivan
It worked this time. Thanks for your support.