I'm currently doing PCR for my study. I used heterologous internal control which I need to include plasmid (Vibrio Cholerae HemM Gene) in each samples. But after I've run about 2 to 3 times pcr, with same samples, the internal control band did not appear with same intensity. Does it normal or it should be only a slight different from each run? Or is it from the gel electrophoresis problem?
Hi Aisha,
I would not be so sure about same PCR conditions, there is always something different in each experiment. (Even though you think you are using same volume, your pipette is probably not getting exactly same volume.)
I could not understand, did you run a gel for each PCR run, or you collect the samples and run all of them in the same gel? Because storing PCR samples may also be problematic. If you run a different gel for each PCR, then again, you can not expect them to have same intensity. (again, conditions! always different!)
Also, you might lose some volume while loading samples into the wells.
So, you can run your experiments in triplicate, and then quantify your gel image with ImageJ (or something similar).
Your gel image (or intensity) could be slightly different but that does not have to mean something is wrong. (probably not) (It is just Wetlab and it hates us all.)
Good luck!
Elmas
All point that Elmasnur Yilmaz mentioned is right, but I need answer of my question.
If you wanna check con of your PCR product, Why you do not use a nano drop for i.e?
Alireza Mordadi I might be wrong but I think she did not mean concentration.
I guess she has to normalize her study/data by using an internal control. (So that's why she wants control bands with same intensity, just a quick guess) (but that's not possible, they will be always slightly different.)
Also, she can not use Nanodrop for PCR product directly because nanodrop measures everything in the PCR mix (primers, template DNA, free dNTPs, primers). She can purify/ or clean up the PCR product but still it is not sufficient. (Also, you will lose your sample a little bit.)
Because you have to be sure you amplified the correct position, you should run on the gel. (You can observe nonspecific amplification on the gel.)
I am just speculating and trying to understand. Of course, I can not be sure.
Best,
Elmas
Elmasnur Yilmaz
I know that all of you said, just wondering of question!!!!
I think she mean some band appear thick and other thin?
Alireza Mordadi Elmasnur Yilmaz
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