PCR triplicates versus one reaction - differences? (Mar/21/2011 )
What is the difference between, for example if i set up a PCR reaction of sample A versus setting up triplicate PCR reactions of sample A then pooling the triplicates together? Is the amount of DNA in the triplicate pool higher (in ng) than just that in the one PCR reaction?
Assuming you are using the same identical sample for every PCR reaction, there is no difference between one large reaction and the same volume reaction set up as three replicates. Having said that, you must consider the fact that a PCR reaction that is 150 ul in volume will behave quite differently from a PCR reaction that is only 50 ul. The larger volume keeps the molecules in the PCR reaction much further away from each other than simply 3 times more separate, so smaller volume reactions tend to be significantly more efficient than larger ones. In other words, you will likely get more PCR product from three 50 ul reactions than from on 150 ul reaction, even if you use the same total amount of DNA.
I don't believe this volume effect at all. There might be some effect, but it would be due to the faster ramping rate of heating and cooling in the cycler, not something mysterious about molecules being closer or further. That's just nonsense.
I am mostly unclear based on what someone was telling me today. here is a clearer example:
they said if they wanted to have ~1ug of sample A in their 20ul PCR reaction, they wouldn't just put 1ug of sample A into the reaction but rather split the reactions into triplicates with 300ng of sample A, then pool triplicates and this person said they will have a 1ug pool. i think they basically don't want to put in 1ug of sample A into the 20ul because it will be too much starting dna, which is understandable as pcr may not work very well with that high starting amount of dna in pcr (it will oversaturate). but is their method correct and is there any other way to do carry this out, to amplify 1ug of sample A in 20ul PCR reaction? I was thinking just reduce number of pcr cycles but any other ways?
IMHO, in the case you mentioned, to do 3 tubes of 300ng in 20ul, is more or less the same effect of doing 1 tube of 900ng in 60ul reaction, everything just scale up X3. (provided your PCR tube had good thermal transfer, no leakage and ramping of your PCR machine is good)
If you compare 300ng in 20ul with 900ng in 20ul, yes, the starting dna might inhibits some PCR reaction.
hope this clears up.
so 3 tubes of 300ng in 20ul is same as 900ng in 60ul...ok so you can increase the amount of dna by splitting up into 3 tubes but the concentrations will stay the same then?
what about 900ng in 20ul compared to pooled triplicate of 3 tubes of 300ng in 20ul? this would be different yes? i worked it out as 900ng/20ul = 45ng/ul and 900ng/60ul = 20ng/ul. is that the right way i'm looking at it? thanks.
adrian kohsf on Tue Mar 22 03:23:53 2011 said:
If you compare 300ng in 20ul with 900ng in 20ul, yes, the starting dna might inhibits some PCR reaction.
Again, How does more DNA in a tube inhibit a chain reaction????
gt_ameya on Tue Mar 22 11:52:10 2011 said:
adrian kohsf on Tue Mar 22 03:23:53 2011 said:
If you compare 300ng in 20ul with 900ng in 20ul, yes, the starting dna might inhibits some PCR reaction.
Again, How does more DNA in a tube inhibit a chain reaction????
Well, it might, if the concentration is consider high.
The concentration "high" is not clearly defined, however for my experience, you can say the DNA template is concentration "high" means when you load genomic DNA into the agarose well and run the gel, you can see the wells looks "bright" under EtBr...that's just my own definition, not peer reviewed...
Refer links below for explanation regarding the concentration issue
http://www.mbi.ufl.edu/~rowland/protocols/pcr.htm
http://www.caister.com/molecular-biology-blog/2008/10/pcr-troubleshooting-template-dna.html
molbio1234 on Tue Mar 22 03:06:14 2011 said:
I am mostly unclear based on what someone was telling me today. here is a clearer example:
they said if they wanted to have ~1ug of sample A in their 20ul PCR reaction, they wouldn't just put 1ug of sample A into the reaction but rather split the reactions into triplicates with 300ng of sample A, then pool triplicates and this person said they will have a 1ug pool. i think they basically don't want to put in 1ug of sample A into the 20ul because it will be too much starting dna, which is understandable as pcr may not work very well with that high starting amount of dna in pcr (it will oversaturate). but is their method correct and is there any other way to do carry this out, to amplify 1ug of sample A in 20ul PCR reaction? I was thinking just reduce number of pcr cycles but any other ways?
If you're afraid that 1ug/20ul would inhibit, you could also do one amp in 100ul (basically, you dilute your DNA without decreasing the amount of starting template). Then when you purify the PCR product with a Qiaquick column for ex, you can concentrate by using an elution volume <100ul.
But in your case, the amount of cycles matter as well. Remember that your curve will reach a plateau. I think that if you do enough cycles (enough to reach the plateau), 3 times 300ng would actually give more DNA that 1 time 1ug (if you don't dilute your primers and dNTP by 3).
i don't understand how you can dilute the dna without decreasing the starting template amount. can you give some examples.