All the pictures I have uploaded are gel image of genotyping. What I don't understand is, most of the time, I get very very faint second band, and I assumed it's probably a problem with primer design.
However, I got a really nice and clear band 2 days ago. Looking at the latest result, I believe primers are working fine.
Recipes and protocols were all identical. The only difference is number of samples I ran was small for the clear bands compared to others.
While writing this question, one thing comes in my mind. Could it be a problem that happened during making the mastermix? But I do vortex quite thoroughly...
What could be the resason why I get faint bands and sometimes get a clear bands?
Thank you Navonil Gogoi . But why could same number of PCR cycles give out different band intensity? Could it be that one primer is just more proficient than the other one, thus more product?
Any error in the mastermix would have affected all reactions equally. If the mastermix looks clear without any preciptates, then there wont be a problem like this. Unless there is foreign DNA contamination (most likely your own) in the reactions where you get the second band, the issue could have something to do with the primer.
1. Do you see changes in this second band's intensity if you do a gradient PCR where you vary the annealing temperature ? If you do see changes in the intensity then the primer pairs are not specific to your GOI and are binding to another site in your template.
2. Does the band not appear if you add 5% DMSO to your reactions ? If so then your primer is forming secondary structures at certain conditions.
Your best best is by varying the annealing time and temperature. By reducing the time or increasing the temperature, you can reduce unwanted secondary interactions during annealing. You will have to test by gradient PCR to be sure.
The amplification of unwanted bands can also happen if you use the product of one PCR reaction as the template for another.
Lastly, you can reduce the primer concentration in the reaction to 0.1 to 0.2 µM. Also, if possible you can increase primer size to improve its specificity and try to use GC clamps at the 3' end of your primers.
Hello Heeseong
The reason why you are not getting consistent results is because you still have to standardize your PCR conditions. The annealing temperature is the issue, As mentioned by Praveesh please carry out a gradient PCR and fix the annealing temperature. Iif this step does not work then you will have to relook at your primer set. I can see a lot of non-specific binding in your gel pic.
Thank you.
Heeseong Cho In my opinion the primers are working fine if you are getting your desired product. May I know what is the region you are amplifying? See different samples are going to have a different amount of DNA conc. even If you use the same extraction kit. There might be also DNA degradation if there is repeated freeze-thawing of your sample. The amount of sample you are using is less for gel electrophoresis that is also a reason for low band intensity. Add more sample for your gel run you will get a more prominent band. Yes, you can do a gradient PCR to obtain the optimum annealing temperature. you can also reduce the primer conc to reduce non-specific amplification. If you just want to increase the band intensity try increasing the cycle and loading more sample to the gel to get brighter bands on gel.
@Praveesh Valissery and @Malcolm Nobre thank you very much, I will try the gradient PCR and see if I can get any better annealing temperature for the 2nd reverse primer.
@Navonil Gogoi, thank you very much too. I am doing genotyping of TRP C4 strain mice. I am not really sure about the knock out gene, I will have to find the information in the lab on Monday.
The DNA concentration was not diluted to make all of them at same concentration, but I always get somewhere around 18~20ng/ug. The samples were not froze and thawed, they went through DNA extraction to PCR on the same day. The volume I use is 2ul of DNA and 18 of mastermix.
I am also planning to increase the number of cycles as well as gradient PCR! Thank you very much for your help I really appreciate it!
Hello Heeseong Cho Did you get your answer for the faint bands? I am facing the same issue when I run mouse genotyping PCR.
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