I set up a small scale PCR of 20uL to check a set of new reverse primer (RP). I have previously used the same template, buffer, enzyme and other reagents (including same forward primer, FP) under already standardised PCR conditions. But astonishingly I didn't get the desired amplicon (only some primer dimer).
1. I had set one with previous primers (both FP and RP) for which also I didn't get any amplicon. So, the problem isn't the new RP.
2. I checked all my templates and they were fine on gel.
3. Next time I did some changes-
a. 20uL reaction with new reagents (I changed everything and made fresh).
b. 50uL with old reagents.
c. 50uL with new reagents.
This time I got amplicons in all 50uL reactions but not in 20uL reaction with same new reagents.
Although the problem is resolved, I wanted to know why the reaction volume would matter so much? Whatever final concentration I used in 50uL reaction I scaled down to 20uL, so it is not due to unavailability of any reagents I suppose.
Even if all concentrations are the same the total amount if all components differ. Maybe an increaae of Template amount can help.
What are you using for a template? If there is low target DNA concentration in your extract, you can have stochastic sampling effects. The more extract used (e.g. in your 50uL reactions) the more chance of template DNA being added. If this is the case, you may find if you ran five 20uL reactions, only two would produce the desired product.
Dear Stephen, I used both a previously amplified PCR product and a plasmid containing the insert as my template. I have checked the quantity and quality of template DNA in an agarose gel. It is surely not less than 100 ng. As PCR is a sensitive method, it should amplify even if there is a lesser amount of target DNA. I should have got less, but some amplicon then!
you cannot always scale up a pcr reaction. many pcr machines are calibrated for a fixed volume and particular thickness of tube or strip and the pcr temperature s are assumed for this volume. So if your pcr machine is on a fast cycle and assumes 20ul volumes then you increase the volume to 50ul then the reaction volume may not ever reach denaturation temperature before it starts cooling again for the next part of the cycle. Similarly it may not drop to your defined annealing temperature as the increased volume will hold its temperature for longer and may not cool sufficiently. In these cases increase the time at 94 and the time at 60 (or annealing temperature) and it may work. Primer dimer works at all temperatures , of course , as it melts easily and anneals perfectly with the primers
Thanks Paul, I completely agree with your logic. But is it true for scaling down as well? Because in my case, the amplicon was there in a 50uL reaction mix but same was not amplified in 20uL. If the machine is calibrated for a higher vol (50uL) will it not work for lower volumes?
Hi Tias,
It is also true that some pcr machines do not scale down well either. Assume that your vol is 50ul and annealing temp is 60. The machine takes its temperature down to 58 block temperature knowing that the sample temperature will lag behind the block temperature by a known time and then the block comes back up to 60 for the hold time. For fast cycling the block must always be ahead of the sample temperature to speed up heating and cooling.If you have a smaller volume than the reference then heating and cooling are quicker so you can get high temperature denaturation or lower temperature annealing which may lead to dirtier pcrs. This logic applies to most pcr machines which do NOT ask you what volume the pcr is carried out in. Usually such machines give the assumed volume in the instruction manual. Fortunately pcr is quite robust so most of the time small changes do not matter but it may be you have found a template and primer set where it does matter eg high GC would care about a slightly lower denaturation temperature and some primer sets are on the brink of working to help clean up the reaction. If you cover your pcr with oil then at 50ul the volume of oil is smaller compared with the same volume on top of 20ul so the whole sample changes temperature differently.
On the other hand if you do not use oil then the volume change of losing some water into the atmosphere of the strip/tube at high temperatures is small compared with 50ul but losing 5ul ( for instance) of water into the tube from a 20ul volume changes all concentrations appreciably and the increased salt concenrartion may be enough to change the Tm of the primers which might have an effect on the reaction.
All of this is conjecture but I am quite certain that generally in chemistry scaling up or down are not trivial matters
hi Bhuvan,
if you have a pcr that has worked for months and stopped working I think that the most common ( of many) reasons is primer degradation. If your primers are dissolved in water then co2 dissolves from the air and the water becomes acidic. the single stranded primer then ger depurinated and falls apart.. Try new primers and dissolving them in TE at alkaline ph which stops depurination and also stops nuclease activity because the edta chelates out all magnesium ions which are needed for nuclease activity. If you put this question to open forum in researchgate you will get many more and different answers to your problem
Dear Bhuvan,
1. My sample is quite old. More than 8 months.
2. I used 150 ng template concentration for scaling up reaction.
3. No, my positive control for 20uL reaction didn't work.
In your case, as your positive control is working, I don't think reaction volume or PCR reagents is a problem. Only thing that has changed is the template DNA. Use fresh isolate and improve your storage condition for the isolates.
Hope it helps!
OId samples of DNA even if it is in stored in -20 degree for long duration are not good for the amplication by any molecular marker system. The number of bands amplified are lower when compared with the new DNA sample for the same molecular marker.
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