Lately, I have read a paper about using mineral oil as a layer above PCR template to prevent the co-evaporation of DNA during RT-PCR process. However, they said that the when using the mineral oil, the DNA concentration increase during the process. I don't know whether the increase in DNA concentration during PCR reaction is good or bad for DNA amplification and have any kinds of effect on the PCR reaction?
Thank you in advance
I agree with Marwa Hany that it would help to see this paper.. I expect the concentration of dna to increase slightly without oil as the air in the tube can hold more water but the dna concentration seems to me to remain lower if the oil layer stops the water from evaporating so I also would like to see the paper . Be aware also that the oil makes a big difference to the total volume of the pcr reaction mix so temperature profiles will be very different between ,say, a 20ul pcr mix and a pcr mix of 20ul with another30ul of oil covering it which also needs to be heated and cooled. It is also quite difficult to add exactly the same amount of oil to all samples but one advantage of oil is that it makes pcr contamination less likely when removing caps from the pcr tubes/strips because it reduces aerosols
can you send the link of the paper?
All I know is that mineral oil is used to protect the sample from evaporation.
but once you use a thermocycler with heated lid, you can exclude the mineral oil.
I agree with Marwa Hany that it would help to see this paper.. I expect the concentration of dna to increase slightly without oil as the air in the tube can hold more water but the dna concentration seems to me to remain lower if the oil layer stops the water from evaporating so I also would like to see the paper . Be aware also that the oil makes a big difference to the total volume of the pcr reaction mix so temperature profiles will be very different between ,say, a 20ul pcr mix and a pcr mix of 20ul with another30ul of oil covering it which also needs to be heated and cooled. It is also quite difficult to add exactly the same amount of oil to all samples but one advantage of oil is that it makes pcr contamination less likely when removing caps from the pcr tubes/strips because it reduces aerosols
Thank you for your answer, the paper said the most effective amount of mineral oil is 7ul, so is there any remind or adjust need to be made about the pcr sample?
When you use the thermocycler machine I think there is no need of using the mineral oil.
The oil is maintained dna evaporation during several heating temperatures, so, at specific dna concentration or increasing its concentration, there are inevitably amplifications for dna to billion of copeis. That have back point we can measure the original copies
Mineral oil was used long before when we still did not have the current thermocyclers, precisely to avoid evaporation of the samples. I suggest you try without using oil first and see if you get good results. With a thermocycler with a lid that controls the temperature well, you should have no problems. Good luck
The paper does indicate that even with oil overlay there is some evaporation. Also it shows that 30ul of oil gives a very poor efficiency of pcr. Not surprising as the total volume of the reaction and oil is 55ul so times taken to achieve denaturation and get down to annealing temperature will be long and the authors do not indicate that the cycle parameters have changes to compensate for this. If they are right and some evaporation takes place even with oil overlay then there does not seem to be any good reason to use this. It is very clear that NTCs are hugely important and that the post pcr work should be done away from the pcr setup areas as post pcr there are billions more molecules around and an aerosol on opening a tube could be disastrous. I do not think that the template dna concentration happening a few cycles into the reaction makes any difference as the amplimer is increasing exponentially but the amount of amplimer being generated from the template dna is only a linear increase.
All the PCRs I did this way before the heated lid thermocycler appeared I never saw a negative effect on the results, just don't pick up a trace of oil with your PCR product.
Honestly, that paper is pretty shoddy work. They were seeing if the type of seal reduced contamination. What that tells me is that whoever is setting up their reactions is not being careful enough to avoid cross-contaminating their samples. Evaporation is not an issue if you seal your plates properly (lid or film).
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