PCR & nested PCR problems

11 posts / 0 new
Last post
chenga
chenga's picture
PCR & nested PCR problems

I have been stucked with the problem i am going to mention hereunder.

I performed the first round PCR for PCV2. Positive and negative controls were included. Except for the positive control, all samples and negative control were negative by the 1st round PCR. Using this pcr product as the template, nested PCR was performed. Utmost care was taken to avoid any contamination by using one pipettes tips per sample although same pipettes were used for the pcr and nested pcr. My lab doesnt have separate room for PCR and nested PCR. By nested PCR all samples and negative control were positive but the positive control turned out to be negative. The result of negative control by nested PCR seem to indicate that there could have been some contamination post pcr. But what could have caused the positive control that was positive by pcr to be negative by nested PCR?

chenga

 

Ivan Delgado
Ivan Delgado's picture
 

 
Hi chenga,

What you describe could be cause by a number of issues, and contamination is but one of them. 

A positive control turning out to be negative after nested PCR could be cause by a number of issues. One of them could be that the nested PCR was overloaded with DNA. It is possible, although not too likely, that too much DNA could inhibit PCR (in this case the nested PCR). Alternatively it is possible that you simply did not transfer the first PCR to the nested PCR. How many replicates did you perform for your positive control? If you performed two or three replicates, did all become negative after the nested PCR? There are times when we are 100% sure we added the template DNA, only to realize that we did not. 

Hope this helped. 

Ivan

chenga
chenga's picture
Dear Ivan,

Dear Ivan,
Thank you very much. Your suggestions were useful.
I perfomed 3 replicates and all the results came out to be same.
I didnt check the actual DNA titers of the positive control sample. I will just check it with different dilutions and see if DNA overload is the main reason for my result.

Anyway thank you for your suggestion.

Chenga

wizard
wizard's picture
Hi Chenga,

Hi Chenga,

I totally agree with Ivan it is most likely to be due to the DNA concentration - did you dilute the product of the PCR before use it as a template for the nested PCR? I would suggest to dilute at least 1:10 or 1:100!

And just have a closer look to you nested primer pair - it happens quite fast to mix up primer...

Good luck
 
Wiz

chenga
chenga's picture
Dear Ivan and wizard,

Dear Ivan and wizard,
thank u for ur sugestion.
i diluted my DNA sample to 1:100.  The result was wonderful.

Thanks.

chenga

wizard
wizard's picture
 Hello Chenga,

 Hello Chenga,

congrats!!! Its nice if problems are solved, isn't it?! Good look for future work!

wiz

baroon
baroon's picture
 dear chenga 

 dear chenga 
how can you overcome contamination that result in negative control of nested PCR to become positive? 

Nikail Collins
Nikail Collins's picture
contamination of PCR template

Hi,I always use a DNAse removal product to decontaminate work areas when working with DNA. 

Ivan Delgado
Ivan Delgado's picture
Test

This is just a test

sachin shetty
sachin shetty's picture
nested pcr problem

I did nested pcr for beta thalassemia, included positive and negative control. Except for the positive control, all samples and negative control were negative by the 1st round PCR. Using this pcr product as the template, nested PCR was performed. result was all samples and negative control were positive incuding positive control. negative control of pcr2 is negative. But what could have caused the negative control that was negative by pcr to be positve by nested PCR?

Ivan Delgado
Ivan Delgado's picture
Negative control

There are a number of reasons why the second PCR would have given a positive signal for the negative control: 1. Contamination. Somehow you got some DNA into the negative control. 2. Non-specific amplification. The second PCR is actually amplifying something else other than what it was designed to amplify. You should repeat the experiment at least one more time to see if you can repeat these results. If you get the same results you may have to redesign your primers.