Primer working with some cDNA samples and not other - (May/28/2015 )

Hello all!

I do conventional RT-PCR of five genes in different cDNA samples. Sometimes I see that while all five genes worked perfectly fine with one sample cDNA, one gene will not amplify at all in another sample (but the other 4 genes will work out fine for that sample).

So for example:

- genes A, B, C, D and E work perfectly fine for cDNA #1

- genes A, B, D and E will work fine with cDNA #2, but all I get are primer dimers of C.

Can anyone tell me why this is happening?

Thank you!

Sakumi

Sakumi on Thu May 28 21:15:24 2015 said:

Hello all!

 

 

I do conventional RT-PCR of five genes in different cDNA samples. Sometimes I see that while all five genes worked perfectly fine with one sample cDNA, one gene will not amplify at all in another sample (but the other 4 genes will work out fine for that sample).

 

So for example:

- genes A, B, C, D and E work perfectly fine for cDNA #1

- genes A, B, D and E will work fine with cDNA #2, but all I get are primer dimers of C.

 

Can anyone tell me why this is happening?

 

Thank you!

 

Sakumi

What do you mean by working fine? Do you mean your Ct values for some samples are undetermined?

I do conventional end point PCR.

So I will do 5 genes using the same cDNA. Four of the genes will give bands of the right size, but one will just end up as dimers.