Hi dear colleagues,
I am amplifying several fragments between 3500 and 7500 bps from cDNA using fusion polymerase, following the provided protocol for mix preparation and PCR program. I managed to amplify fragments with the expect band size. More or less 24h later, in order to purify the bands, I scaled-up the reaction and guess what? No bands! Only a hight molecular weight band appeared after long exposure time, which I suspect that it corresponds to the cDNA template. To exclude preparation errors, I repeated the same protocol and the result was the same. What can go wrong in 24h? Phusion was at RT for few seconds while pipetting and then kept on ice block for a minute or 2. Primers thawed at RT and remained at RT for a few minutes while preparing the PCR mix, as well as the dNPTs. Were these minutes on hold at RT sufficient to degrade the primers or other solutions? cDNA doesn't seem degraded as no smear appears at the gel after prolonged exposure time. Neither primer dimers... Some good tips would be deeply appreciated!
Thanks in advance,
Luisa
When you scaled up the reaction it may have had a large effect on the pcr temperatures and the time ramping between those temperatures. For instance if you changed from 10ul to 100ul total volume then the 100ul will keep its heat and when coooling to the annealing temperature the block may reach Ta but the reagent mix will still be hotter than that so primer annealing will be less. Equally the heating step to 94 the block reaches 94 but the reagents lag behind the block and may not reach 94 before the block is cooling again for the next cycle. Instead of scaling up stick with the working assay and run 10 tubes of small volumes not 1 tube of large volume. You can always pool the product and run your checker gel on the pooled product not on every small tube
Thank you very much Paul Rutland. I scaled-up from 20uL reaction to 2 tubes with 50uL reaction (in 24h time)... Should 2.5x volume increase enough to explain this delay in temperatures? At my second unsuccessful try of the day, I used 20uL reaction again, so it should have worked, right? Still, what you say makes sense to me because it has happened in the past that my bands of 20uL reactions were always stronger than the 50uL reactions (sometimes no bands were present)....
Thanks again Paul,
Best,
Luisa
I think that a 2.5 fold change will make a difference. Concentrations change and rates of chemical reaction are concentration dependent. Another factor is that most pcr machines ( especially old ones) are calibrated at a fixed volume and changes to the reaction volume may cause undershoot and overshoot in temperatures as the change in volume to larger volumes causes a temperature inertia in cycling. Usually overshoot on cooling and undershoot on heating for larger volumes and the opposite when making volumes smaller ( reaction works but non specific bands appear). If you have a pcr machine that allows you to set the reaction volume then the manufacturer has taken these changes into account and reactions work over a wider volume range
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