8 replies to this topic

[niclam]

Hello Everyone,

New to the forum here. I've come in search of help to find out why all of a sudden all my qRT-PCRs look like this. I've been using the same primers, same mix, etc, etc. Just now the amplification is horrible and maybe there's high background? I'm not at all knowledgeable about real-time, I just let the machine do all the thinking for me. I've repeated it 2 more times and it still looks like this. Please help!!

Attached Thumbnails

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[nightingale]

Hello Niclam,

it seems you haven't turned off the extra channels in the result page ...
i mean ...
u are in this window showing not only your samples ...
am i right ?

[niclam]

nightingale, on 04 January 2011 - 01:35 PM, said:

Hello Niclam,

it seems you haven't turned off the extra channels in the result page ...
i mean ...
u are in this window showing not only your samples ...
am i right ?

Thanks for the reply. The curves shown are my all my samples plus the standard curves using genomic DNA

[tea-test]

To be honest, the curves look quite normal to me if you are using SYBRgreen, but I don't know how they looked like before. It seems that the y-axis is in linear scale, maybe change to log-scale and then look again.

[ElHo]

tea-test, on 05 January 2011 - 01:57 AM, said:

To be honest, the curves look quite normal to me if you are using SYBRgreen, but I don't know how they looked like before. It seems that the y-axis is in linear scale, maybe change to log-scale and then look again.

I second this. The curves look okay. Must be something about the settings that someone might have changed. So don`t worry.

[nightingale]

as long as the curves are of your samples, so :
i ,too, third tea-test & EIHo ...

just thought that you are seeing those curves that are not real in reality as you have kindly wondered about a high background ...

[niclam]

It is already plotted on log graph already. Previous runs have a lower baseline (see attached).

Attached Thumbnails

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Edited by niclam, 05 January 2011 - 10:29 AM.

[ElHo]

Looks like a problem concerning baseline to me. Are there any differences concering baseline settings in your current and previous runs? In my opinion your previous run look more strange than your present one.

[nightingale]

well,
attaching the latter picture, made your challenge clearer ...
at least ... to me !

if i got you right :
you are wondering why you are now elevating the baseline
to a higher level in order to analyse your run.
right ?

if yes,
we once faced such a problem ...
and changing the mastermix vial solved it !
don't ask me, why ...  
we didn't know !!!

although, as just your case :
using the same vial previousely worked fine.

i know expensive, but that was the solution ...  

you may try this :
Adjust baseline correction ie from cycles 3-15 to 3-10 or dilute template between 1:100 and 1:1000
and repeat.
taken from the guide attached.  QPCR_&_QRT-PCR_Troubleshooting_Guide.pdf   76.17K   100 downloads

Good Luck & kindly keep us on track ...

Best Wishes