If going from 25µl --> 50µl interferes with the rxn, then try making two or three 25µl rxns and run them simultaneously to have enough of the desired product.

If going from 25µl --> 50µl interferes with the rxn, then try making two or three 25µl rxns and run them simultaneously to have enough of the desired product.

Sometimes, the PCR is not reproducible in cases of some pipetting mistakes or wrong dilutions. Just prepare everything freshly with care & repeat the reaction either as one 50 micro litre reaction mixture or two 25 micro litre reaction mixtures as desired and it should work. Generally, the PCR machine asks you to enter the reaction volume before the start of the run. Make sure to enter the right volume.

Hope it works,

Best Regards,

Sagar

The other problem, not mentioned here yet, is that termocycler may induce tiny differences in heating the sample with different volume. Smaller volume sample is easier to heat up/cool down during short-term step than a larger volume. I recommend the same what Michael and Sagar - just use separate 25 uL mixtures and combine them after PCR reaction.

HI

Ramping of cycler or thermal transfer of PCR tube or leakage needs to be considered. And if all these things are fine then I would suggest to double all the things except for template.

And when you say it did not work, you need to say what it looks like on gel; may be that could help others to answer well.

Cheers.

Have you considered to enter the volume of your sample on the cycler before you start run? Usually, and after entering the run details, the cycler will ask about the volume of your sample. It is important to enter the correct amount, and not just any number!

Good luck!

Yes,the optimized volume for uniformity in temp variation worked well with 25 microlitre vol in thermo cycler,that may be the reason and variation in temp may not cope with volume greater than 25micr litre ..

Dear, don't need to be worry, some time its happened but i thinks so there may be pipeting error during reaction

Hi dear friend,

don't worry, PCR is complex reaction and same problem occurred sometime, Check the PCR instrument, temperature and control your Sampler pipeting, also download and use of below pdf.

Good Luck

Dear Eswara, I think, nearly everything has been said - first, the temperature of your PCR samples during reaction is of course not directly measured, it is calculated - since heavier objects (larger reaction volumens "m") need more energy ("Q") to reach a certain temperature ("Delta T") than smaller ones: Q = c * m * Delta T, you might get a temperature gradient in your samples (especially when time of constant temperature is short, for example 15 seconds at 95°C) resulting in insufficient conditions. On the other hand, PC-Reactions are quite complicated and concentrations of oligonucleotides and cofactors (especially Mg2+ Ions) have to be optimized within each protocol. So if you need further assistance with optimizing PCR, maybe this document I found on the Internet might help you: http://www.babec.org/files/PCR_2012/PCR_Optimization_Student_Guide_2012.pdf or http://www.babec.org/node/13 .

Regards

HI,

I also suffered from same problem couple of years ago. Later I found that it was because earlier I was just doubling the volume of each component of PCR and in the process, the final concentration of dNTPs and primers changed. So, I calculated the volume of each component in my reaction according to the concentrations and that worked.

hope this helps.