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Weird qPCR curves


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#1 peanut142

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Posted 17 January 2012 - 07:11 PM

Hey guys,

I have been doing qPCR for a while and everything was fine until last week. I observed this weird curve. Basically the SYBR signals dropped around cycle 30 for all my wells. I have never seen this before. I checked the machine and it was fine. I repeated the same experiment, still saw the same weird curve. The dissociation curve looks normal for both runs.

I can't figure out what went wrong. Has anyone seen similar curves before? Desperate for an answer. Any guess is welcome as well.

Thanks a lot guys!!!!

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#2 peanut142

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Posted 18 January 2012 - 04:51 PM

Does anyone know????? I am desperate!!!

#3 phage434

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Posted 18 January 2012 - 06:06 PM

I'd suggest that your program is doing 30 cycles of normal cycles then something different for the last several cycles. Can we see the program? Another possibility is evaporation of the liquid in the well, but it seems unlikely that it all happens on the same cycle.

#4 peanut142

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Posted 18 January 2012 - 06:14 PM

Hey

Thanks a lot for your reply. My program was the same for 40 cycles. Okay, the overall program is 95oC 10min for 1 cycle, then 95oC 30s & 48oC 30s (40 cycles), and 1 cycle of 95oC 1min, 55oC 30s and 95oC 30s.

The liquid in the well was not evaporated because I checked for that.
It is very weird because it does not occur every single time. Sometimes it happens and sometimes it does not. It is driving me crazy as sometimes I have to pray before I do my experiments.....




I'd suggest that your program is doing 30 cycles of normal cycles then something different for the last several cycles. Can we see the program? Another possibility is evaporation of the liquid in the well, but it seems unlikely that it all happens on the same cycle.



#5 phage434

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Posted 18 January 2012 - 06:45 PM

There is no extension ?? I'm guessing you just omitted it. I have no clue what the last cycle of 95/55/95 is supposed to do. Are you using a heated lid? Evaporation within the tube could cause this. What is your reaction volume?

#6 peanut142

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Posted 18 January 2012 - 06:50 PM

There is no extension ?? I'm guessing you just omitted it. I have no clue what the last cycle of 95/55/95 is supposed to do. Are you using a heated lid? Evaporation within the tube could cause this. What is your reaction volume?



Yes, I am using a heated lid. We use stratagene mx3000p for qPCR, which should have heated lid. Also I did not see evaporation around cycle 30 of my qPCR (I opened the lid and checked). My reaction volume is 20ul.

#7 phage434

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Posted 18 January 2012 - 06:57 PM

You didn't answer the question about extension time. What is the length of your amplicon?

#8 peanut142

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Posted 18 January 2012 - 07:11 PM

I don't have any extension time. My product is only 51bp long, so both annealing and extension occur within 30s at 48oC. I know you may think that I won't get any product that way, but it works really well in my situation.

Edited by peanut142, 18 January 2012 - 07:12 PM.


#9 peanut142

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Posted 19 January 2012 - 11:04 AM

[Trof: This is a known phenomenon, it's caused by the machine inability to process the high signal or something. There is actually no decrease in DNA, don't worry.
I'm trying to find the brochure where I read this, it was Roche I think.]


Hey, Trof, thanks for your suggestion. The weird curve does not affect wells with high copy number of candidate DNA, but messes up those which have low copy number, as the curve does not come up at the right cycle. Could you please share me the link if you find the brochure? I cannot find it myself.

Many thanks again!

Edited by peanut142, 19 January 2012 - 11:05 AM.


#10 Trof

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Posted 19 January 2012 - 11:23 AM

I deleted my post after a moment, because I looked at the picture again and better (busy moments between cloning and sequencing, sorry) and I'm not sure it's this case. That looks usually a bit different when all the samples are in plateau.

This looks almost like kind of a machine error. But that would happen randomly in any experiment. If it's only in a particular assay, it's weird. Did you try to check it on gel later? SYBR shows decrease in DNA, but you could compare on gel if it's true. If not, then it's problem in SYBR (degrading?) or fluorescence measurement. I would probably try to send this to Stratagene support too, what they think.

phage434: 95/55/95 would be melting analysis I guess, there is missing the ramp from 55 to 95.

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#11 peanut142

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Posted 19 January 2012 - 11:33 AM

I deleted my post after a moment, because I looked at the picture again and better (busy moments between cloning and sequencing, sorry) and I'm not sure it's this case. That looks usually a bit different when all the samples are in plateau.

This looks almost like kind of a machine error. But that would happen randomly in any experiment. If it's only in a particular assay, it's weird. Did you try to check it on gel later? SYBR shows decrease in DNA, but you could compare on gel if it's true. If not, then it's problem in SYBR (degrading?) or fluorescence measurement. I would probably try to send this to Stratagene support too, what they think.

phage434: 95/55/95 would be melting analysis I guess, there is missing the ramp from 55 to 95.


Thanks again. I will email our stratgene rep here.

#12 Adrian K

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Posted 21 January 2012 - 04:33 PM

Signal dropped on cycle 28 for all sample. Unless someone sabotage your work, else is a machine failure, or there is some problem with the software algorithm.
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