11 replies to this topic

[smndtupidisaftr]

Funny problem I've been having recently. I'll amplify a PCR product of about 300 bp, and when I run it out on a gel, the negative control shows a band of the same size and intensity of my experimental lanes. I've changed the water, changed the enzyme, changed the dNTP, changed everything. Still it keeps happening. I would think maybe I missed contamination somewhere, but it's confounding me that the negative control is as bright as the other lanes. That's the part that I can't figure out. What's going on?

[Tabaluga]

Hm, i think that since it's that bright it's unlikely to be a contamination by aerosol. You said you changed every reagent... Do you change your gloves or at least take great care when opening the tubes ? and the tips ?

[smndtupidisaftr]

I do take great care with the tips. With the gloves, I'm sure something might happen there. But would contamination from the gloves be enough to cause a band as bright as the others? I'm considering the fact that the template is the last thing I add. The negative control is done and sealed by the time I even touch the template.

I was thinking that using the PCR strip tubes may be an issue. Could using individual PCR tubes make a difference?

Edited by smndtupidisaftr, 26 February 2013 - 03:50 PM.

[bob1]

Have you considered that it might be more contamination in more than one reagent, unless you mean by "changed everything" you mean that you changed all the reagents at the same time.

It could also be that there is contamination in your primer stocks (not the working dilutions).

[smndtupidisaftr]

bob1, on 26 February 2013 - 04:04 PM, said:

Have you considered that it might be more contamination in more than one reagent, unless you mean by "changed everything" you mean that you changed all the reagents at the same time.

It could also be that there is contamination in your primer stocks (not the working dilutions).

I don't think it's the primer stocks. When I amplified something else with the same primers, I got the correct product of the correct size again. So it looks like each time it's amplifying the right thing, but somehow it keeps getting into the negative control.

[Tabaluga]

Does the same 300 bp bright band appear when you do other PCRs with different primers ?

[smndtupidisaftr]

Tabaluga, on 26 February 2013 - 04:09 PM, said:

Does the same 300 bp bright band appear when you do other PCRs with different primers ?

No. When the expected product is 300 bp, a 300 bp product shows up in the negative control. In this other amplification, the expected product is 400 bp, and a 400 bp product shows up in the negative control.

[bob1]

Is the template for the 400 and 300 bp products the same?

[smndtupidisaftr]

bob1, on 26 February 2013 - 06:47 PM, said:

Is the template for the 400 and 300 bp products the same?

It is the same with an addition to it.

[bob1]

There's the problem, some of your reagents, probably more than one, and/or equipment have been contaminated with this template.  Make sure that you are using tubes straight out of the bag, and that you either use filter tips straight from the box (i.e. don't fill boxes yourself and autoclave, autoclaves are very dirty) or make sure that you extensively clean your pipettors and use pre-boxed normal tips.

If you do a lot of PCR it is a good idea to have pipettes that you use only for setting up the reactions, that never come near the template or any PCR products.

[Ensyeh]

Hello Member!
I dont know if you resolved your problem or no, but my idea is a contamination in your loading dye.

[bob1]

Ensyeh, on 05 March 2013 - 02:14 AM, said:

Hello Member!
I dont know if you resolved your problem or no, but my idea is a contamination in your loading dye.

A good idea, but if this was the case smndtupidsaftr would have seen both bands in the gel, no matter which product they were running.