I am trying to check the polymorphisms of a particular gene from human DNA samples. A set of primer in a published publication allowed the amplification of the DNA hence permitting the analysis of three of the numerous polymorphisms of the said gene.
I used Pfu DNA polymerase but my PCR products gives double/ multiple bands when viewed under agarose gel electrophoresis and this results in mixed peak when the products obtained are sequenced.
Any suggestion on what l can do to get a single clear band and also sequenced results with no noise and mixed peak would be highly appreciated.
Decease the template DNA and Taq enzyme amount and select the suitbale the appropriate annealing temperature
In my opinion, the primers used in your experiments are maybe not specific (only for your region of interest). Then you should identify a more adequate and/or more specific primer oligo.
Another idea is that you should try to advance performance of our PCR by adjusting - especially - annealing temperature (something above 60°C), maybe by performing a gradient PCR.
This should help to avoid any technical problems maybe mirrored by different apparatures used in your lab vs the lab done before (see corresponding publication).
I would guess that you would get less Extra banding if you dropped your cycle number down. Definitely try out the gradient options as previously suggested.
Also consider the possibility that you are getting multiple bands because you have multiple templates. Contamination with your own DNA must be rigorously excluded. Are your blanks blank?
You may want to consider that you do actually have effectively one band from the pcr. Since you are looking for polymorphisms then it is likely that they do exist and that the larger band is really a heteroduplex band of mixed polymorphisms . Heteroduplices do always run slower than the homozygote band due to the single stranded bubble in the double stranded amplimer. .You could sequence each band ( or even the 2 bands together) to get a feel for whether they are almost exactly the same thing. If the bands are different amplimers consider nested primers for a re amplification or touch down pcr as a means of cleaning up the product
Hi, Joshua Adekunle Babalola.
Could you tell us what company enzyme you're using? For me, by your pics with your programming, seems to be the GoTaq from Promega but you're saying you used Pfu enzyme. So, I am not quite sure ...
So, initially, in my opinion, you should change a few details in your PCR reaction programming and test it again.
1 - Activation of the Pfu (or GoTaq in the case you tested both) enzyme only requires 1 - 2 min at 95ºC (you're doing 5 min. I'd diminish it to 2 min because, at the company datasheet, they say "3. Place the reactions in a thermal cycler that has been preheated to 95°C. We recommend heating the samples at 95°C for 2 minutes to ensure that the target DNA is completely denatured. Incubation longer than 2 minutes at 95°C is unnecessary and may reduce the yield."). I guess it may be occurring in your reaction, at least, partially;
2 - The denaturation usually works well with only 30 seconds (I am used to do PCR for 3KB products of viruses and they usually gets better than the one you are presenting here). If you don't feel ok with 30 secs, use 45 seconds in the denaturing step instead.
3 - Check your primers once more. If you consider they are fine, go on.
4 - I'd rather do extension with 74ºC for 6 min (as the company says 2 minutes for every 1Kb and you have 3Kb. Ok? and temperature for extension from 72 - 74ºC - maybe 73ºC).
5 - Lastly, you're speaking on volumes of reagents instead of masses or units. So, what DNA amount you're using for your PCR? Usually, for genotyping 50ng are enough. Although it will depend on the quality and integrity of it. Secondly, how much enzyme? The company recommends 1,25U for every 50uL of reaction. If you're doing 25uL reactions, you should be using half of it. Is it what you're doing? So, for both parameters here, I would recommend you not to add an amount higher than that, because more is not always better (In PCR, may be worst).
Hope it helps.
Please, let us know If our ideas helped you or not. Ok?
All the best.
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publication