PCR reaction without template gives a product - (Oct/11/2010 )

Hello everyone,

I really hope that someone has some advice cause I'm going crazy here...

Attached is a picture of what my PCR used to look like:


..and what it looks like now

Before I would see the primer dimers, and two lanes above, the topmost being the product, the lower one being the single stranded oligo that did not get amplified.

If the lane after the marker in my second picture is lane 1, then this is how it goes:
1- only foward primer, no reverse
2- only reverse primer, no forward
3- normal reaction with everything
4- no Taq
5- no primers at all
6- no template - this is funny - how come I get a product?? my primers are both aboud 20 bases long, so that's not a primer dimer I see
7- no template, forward primer only
8- no template, reverse primer only

I have repeated this many times, reordered primer, working clean, and yet I still get the same result. It must come from the primers, but I never had it before, and now all of the sudden I have it?? I'm getting really frustrated here...

I appreciate any help that I can get.

Khia

It means that one of your reagents is contaminated with template. It could be in anything, what makes you think it is the primers? Especially since you have ordered a new set. Could be in the buffer, water, dNTPs...

Khia on Mon Oct 11 12:56:08 2010 said:

Just to make sure, were all these (1-8)prepared at one time and amplified or did u do it with different sets of reaction ingredients.
Cause if 6, gives you a band (due to some contamination), then 7 and 8 should also give bands (due to the same contamination).
And did you also say that the same phenomenon is seen with the newly ordered primers as well???

It's not a contamination, I have replaced all the reagents, and the primers.

Gt-ameya, they were all prepared at the same time, from the same master mix. I guess it's not a contamination but that my primers, when combined, and with Taq, simply form a product by themselves. I think I'll design new primers...

Khia on Tue Oct 12 08:26:25 2010 said:

It can be the contamination (eg. fungal growth) from water that is getting amplified with your primers. Because you have the amplification only when you use both the primers. We had similar fungal contamination in our MiliQ unit and we used to get similar amplification in no template blank. Try one PCR with water from different source
Good luck

All of the reagents? Including your enzyme? The contamination could also possibly be coming from your pipette. You could try setting your reaction up using new reagents, a different aliquot of enzyme and someone elses pipette.

"but that my primers, when combined, and with Taq, simply form a product by themselves" This is impossible.

Khia on Tue Oct 12 08:26:25 2010 said:

I dont think there is any contamination in your ingredients. But primers combining with Taq to form a mystery product does not seem possible as well. Its probably your pipette then or your MilliQ/ mol. grade water is not upto the mark. YOu should try changing these one at a time.

Good luck....