PCR troubleshoot - (Aug/31/2014 )

Hello, Im a graduate student. Now, I working on part of PCR lab. The problem is I use the condition that my adviser was use and it give a good result with a thick band of PCR product but last two week I perform the experiment with the same protocol as previous and I got the faint band. So help me please.

Most likely one of the components has gone off, probably the dNTPs. Make fresh aliquots of the dNTPs, the primers and your template DNA and test them.

Thank you a lot,

However, now I use new master mix and also order a new set of primer.

When I use 25 ul/reaction and load 5 ul into gel it give a good band but when I make 100 ul/reaction band is faint than 25 ul/reaction.

help me please.

So are the concentrations of reagents different between the two volumes? How much DNA do you add to each? Are the cycling conditions the same? Are you mixing properly?

keep in mind that the thermal characteristics of 100ul are different from those of 25ul. you may want to try making 4 X 25ul reactions and combining them after cycling.

mdfenko on Mon Sep 8 11:46:47 2014 said:

keep in mind that the thermal characteristics of 100ul are different from those of 25ul. you may want to try making 4 X 25ul reactions and combining them after cycling.

Yes, this what I would do, if I wanted more product.

Recently, when I sent a sequencing provider multiple tubes of the sample sample , so that they could be used in case additional sample was required, he reverted back saying that " PCR IN EACH TUBE WORKS DIFFERENTLY and products from different tubes should not be mixed". 

Thanks a lot everyone, it has been a long time that Im not log in this site.

Now, Im already solved this problem.   

And tmr I will have thesis defense.