I did COLD-PCR for JAK2 wildtype and mutant samples. The previous results were okay, the peaks were around 76-77 celcius. Now the peaks are around 77-78, I wonder what happened. My samples are kept in -80 celcius freezer, I have tapped and spinned down them and my master mix before running them on RotorGene.
As the other respondents have indicated, this is likely to be an effect of minor changes in buffer conditions, possibly due to inaccurate pipetting. The possible reasons are many. A leaky seal in one of your pipettes is one possibility.
Note, however, that the PCR reaction itself will change the buffer conditions, due to the release of inorganic phosphate from the dNTPs. This causes the Tm to drift slightly as the concentration of amplicon (and thus Pi) increases.
As long as the Tm's for your samples match those of your controls, this should not be a big problem, but keep an eye on it, as it might be a sign of other problems on the horizon.
Hi,
where you kept master mix?
4oC or -80
is there is chance to dehydrate any of them, and increase in salt concentration.
at the time of reaction setting also room temperature matters, if buffer dehydrate their effect will multiply.
may be...
ok
thank you
Dear Juli
To ensure accurate reporting you should follow MIQE standards. Part of that would mean running controls and a standard curve in each experimental run. Also running controls and query samples on the same plate to control for experimental bias. If you do this any deviation in a relative quantification (std curve or reference samples or both) will be negated. This is important for publication.
Good luck!
As the other respondents have indicated, this is likely to be an effect of minor changes in buffer conditions, possibly due to inaccurate pipetting. The possible reasons are many. A leaky seal in one of your pipettes is one possibility.
Note, however, that the PCR reaction itself will change the buffer conditions, due to the release of inorganic phosphate from the dNTPs. This causes the Tm to drift slightly as the concentration of amplicon (and thus Pi) increases.
As long as the Tm's for your samples match those of your controls, this should not be a big problem, but keep an eye on it, as it might be a sign of other problems on the horizon.
Also to consider is inter-well variation of your instrument as this may result in errors such as the one you have described. I've had very different melting peaks for the same sample run across all 96 wells of my qPCR machine, now I know which wells to avoid.
- Badges
- Science method