What did you mean of same intensity ?

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

If you perform 25 indepent PCR's with your internal control in the matrix you perform your experients (but without the specific primers) you may find much variation between individual PCR's, due to pipetting issues, variations in individual efficiencies and slight differences in sample volumes. A 5% lesser volume of your sample may easily lead to 3-4 logs differences in output concentration (i.e. dfferent band width in your electrophoresis). What you first need to do is inventorise the inter-experiment variabiity of your internal control WITHOUT your specific primers but with the sample matrix (!) with, say 15 individual assays performed on 15 different days. Then do the same in the duplex with your positive control sample. Herewith you can judge the stability of your duplex system. If the variability remains, you at least kow that it's an inheritant property of your sample and you can incorporate this issue in your quality control procedure.

It may also be, that you use your primers of your internal control at a critical concentration so that slight differences in reaction conditons will have a large impact at the PCR efficiency.

In all cases, It would be much better to perform qPCR. Then you can see what happens during PCR.