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Gradient PCR works, but single temperature does not! HELP!!


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#1 Doggie Science

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Posted 13 July 2010 - 01:15 PM

Hello Everyone,

This is realy weird to me: our gradient PCR works, but once we select a optimized temperature and redo the reaction it does not!!

This has been happening with different primer sets, all ~500bp aplicons.

Here is what we have tryed to change so far:

- Have used an old machine and a new BioRad C1000 - problem persists
- Have changed all reagents to fresh reagents - problem persists
- At this point we are keeping the gradient reactions and the single temp reactions at the same volume (25ul)- problem persists
- Changed Taq brands -problem persits
- Mg 2+ gradient - problem persists
- rediluted primers - problem persists

At first we just thought our old machine was bad and the gradient temperatures and the single temperatures were off, but now we do not know what to think. We get vibrant specific bands for the gradient PCRs (sometimes with one non-specific).

Please let me know if you have any ideas!!

Thanks

#2 Adrian K

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Posted 13 July 2010 - 05:54 PM

Are you aware that in gradient PCR, all the temperature ramping rate is different for each well?

Template degraded? Primers degraded? Water contaminated?
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#3 HomeBrew

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Posted 13 July 2010 - 08:21 PM

Seems the one thing you haven't tried is new primers, other than rediluting them. Maybe a re-designed set of primers would help?

What are the Tm's of the primers you're using? I could see this situation occurring if the Tm's are far apart...

#4 Doggie Science

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Posted 14 July 2010 - 09:27 AM

Hi guys,

Thanks for the imput. We have changed the water, template (genomic DNA), and everything else other than redesigning the primers.... which doesn't make sense since they do work in the gradient. And the primers all have ~58 Tm. And the pairs do not vary by more than 2 degrees.


I was also thinking of the ramping rate... Are you saying that each row ramps differently?? I am going to try and look this on my machine manual... thanks for the idea.

#5 HomeBrew

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Posted 14 July 2010 - 11:08 AM

What cycling conditions are you using for the non-gradient PCR? What is your PCR product size?

#6 Adrian K

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Posted 14 July 2010 - 05:06 PM

Hi guys,

Thanks for the imput. We have changed the water, template (genomic DNA), and everything else other than redesigning the primers.... which doesn't make sense since they do work in the gradient. And the primers all have ~58 Tm. And the pairs do not vary by more than 2 degrees.


I was also thinking of the ramping rate... Are you saying that each row ramps differently?? I am going to try and look this on my machine manual... thanks for the idea.


Yes, when we do gradient each rows ramps differently so that all the rows can achieve the desired temperature together. This only happens in gradient. For my case, I can copy the ramping rate when I look into the machine's log. (I'm using biorad mycycler) I will only concern about ramping when only the gradient works but the normal condition failed.

Perhaps you can try another round of gradient (with your existing primers and pcr condition) to see if the amplification works again, compare with non-gradient. If this second trial does work in gradient (but not working in non-gradient), most probably it had to do with ramping. If is really have to do with ramping, increase the annealing time and extension time will do (by 10-20 seconds each).

Which cycler you are using? amplicon size?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434




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