7 replies to this topic

[Sherrie-Ann]

We are getting an unwanted 100bp band after RT-PCR, with our correct
amplicons and appears in all samples, including a non-template control.
Sometimes it's more prominent in some lanes then others after been ran on a
agarose gel.

Do you have any suggestions as to why we would be getting this? We have
considered that the kit has been contaiminated. Also more than one person
has used the kit and obtained the same results. Also we have used different
different kits, and seen the same results.

[leelee]

My guess is primer dimer??

[Sherrie-Ann]

leelee, on Jun 9 2009, 10:52 AM, said:

My guess is primer dimer??

Well we have thought of that, it's a bit to big for that, and we have checked if they will self anneal. And as mentioned before it's more prominent in some samples then in others and all the samples have their own primer sets.

Thanks for the tip

[gfischer]

If everyone using the kit has the same problem, regardless of sample or primers, it must be reagent contamination.  I'd go ahead and buy a new kit, it's a lot less expensive than your time spent trying to figure this out.  Also check your hood and plates and make sure they're sterile.  Maybe get new plates, or at least resterilize them.  If you can, I'd even try setting up the PCR in a different hood, to see if that makes a difference.

[Sherrie-Ann]

gfischer, on Jun 9 2009, 04:05 PM, said:

If everyone using the kit has the same problem, regardless of sample or primers, it must be reagent contamination.  I'd go ahead and buy a new kit, it's a lot less expensive than your time spent trying to figure this out.  Also check your hood and plates and make sure they're sterile.  Maybe get new plates, or at least resterilize them.  If you can, I'd even try setting up the PCR in a different hood, to see if that makes a difference.

Yes we have considered that, and yes we are getting a new kit, but we got the band with two other kits and 2-Step RT-PCR. The water source is the same however, they have been treated differently. Well we will be able to rule out contaminated kit when our new one arrives.

Thanks for your suggestion

[leelee]

So it would make sense that the water is contaminated then. Or maybe a pipette?? Have you all been using the same pipette to set up your PCRs??

[Sherrie-Ann]

leelee, on Jun 10 2009, 03:54 AM, said:

So it would make sense that the water is contaminated then. Or maybe a pipette?? Have you all been using the same pipette to set up your PCRs??

No we have not! We don't even work in the same labs. Lets hope it's the kit! We are also changing our water to Milli-Q water. So lets hope it was as simlpe as it been the water.

[swanny]

Sherrie-Ann, on Jun 10 2009, 09:08 PM, said:

leelee, on Jun 10 2009, 03:54 AM, said:

So it would make sense that the water is contaminated then. Or maybe a pipette?? Have you all been using the same pipette to set up your PCRs??

No we have not! We don't even work in the same labs. Lets hope it's the kit! We are also changing our water to Milli-Q water. So lets hope it was as simlpe as it been the water.

Simple and very frustrating!