Troubleshooting Primers PCR

[Susan_]

Hi,

I have a problem of no amplification with my primers. The problem is: the PCR give amplification because the primers are well designed but after a while this PCR does not work. The first time that it didn't give amplification I changed all the components included the ADN and still did not work. After I buy new primers the first two amplifications were good and after and then still did not work. My laboratory members also have the same problem.

May be what are the primers unstable or fault of dilute?

The solvent where we dilute the primers is TE ( Tris 0,01 M, EDTA-Na2 0,004 Mm, pH 8).

Thanks

[aelshafey]

Are you amplifying DNA or cDNA? Try to increase your primer concentration in reaction e.g. use 30 pmol of each primer. Dilute your primer with DNAse free water to a final concentration of 20 pmol/microleter, store your stock primer diluted at 100 pmol/microleter. Use filtered tips for your PCR preparation. That is all I have

[davidflora]

I agree with you aelshafey...I think increasing primer solution may be the solution..Just try this susan_

[Susan_]

Originally Posted by aelshafey

Are you amplifying DNA or cDNA? Try to increase your primer concentration in reaction e.g. use 30 pmol of each primer. Dilute your primer with DNAse free water to a final concentration of 20 pmol/microleter, store your stock primer diluted at 100 pmol/microleter. Use filtered tips for your PCR preparation. That is all I have

I 'm amplifying DNA.
Dilute the primers with DNAse fre water is it very common?

Thanks.

[germagno86]

Originally Posted by Susan_

I 'm amplifying DNA.
Dilute the primers with DNAse fre water is it very common?

Thanks.

In my lab we use bidestillate water, sterile.