17 replies to this topic

[molbio1234]

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?

[ivanbio]

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.

[phage434]

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.

[molbio1234]

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?

Edited by molbio1234, 21 March 2011 - 07:13 PM.

[Adrian K]

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.

[molbio1234]

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.

[Ameya P]

adrian kohsf, on 21 March 2011 - 07:23 PM, 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????

[Adrian K]

gt_ameya, on 22 March 2011 - 03:52 AM, said:

adrian kohsf, on 21 March 2011 - 07:23 PM, 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.e...otocols/pcr.htm
http://www.caister.c...mplate-dna.html

[Maddie]

molbio1234, on 21 March 2011 - 07:06 PM, 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).

[molbio1234]

Quote

you could also do one amp in 100ul (basically, you dilute your DNA without decreasing the amount of starting template).

i don't understand how you can dilute the dna without decreasing the starting template amount. can you give some examples.

[Ameya P]

Adrian,

One of the links you posted said this:

9.Excess or insufficient template. Too much template can inhibit PCR by binding all the primers. Too little template, and amplification

may not be detectable. For 25 - 30 cycles, 104 copies of the target sequence are sufficient. [citation needed]

My argument is,  if the template binds all primers, you are off to a very good start.

and 104 copies of target sequence     how does one know that?


Adrian, please note this is a scientific forum, and all data your present, must be peer reviewed  

[Adrian K]

Ameya, I do know that this is a scientific forum.
Peer reviewed.. ok... since I do not have any solid evidence yet, why not I do a reaction and let you be my peer reviewer?  

Now, what about a published book:
http://books.google....epage&q&f=false

Read page 18 to page 19... convinced? If further info, you can contact the author: Michael L. Altshuler

[molbio1234]

adrian, I like your links , am seeing some great book finds I can buy through amazon too

Wondering if you have a book link for this: most PCR guides or troubleshooting manuals describe the generic statement that just having one copy of your target gene (or one copy of DNA) is enough for PCR to amplify it in millions of copies due to the exponential nature of amplification. But I usually read just about one copy. Do you know of any books that talk about low copy number of DNA or target gene in PCR reaction and what happens there, also high copies of DNA or target gene and really go into the details of these PCR reactions and what is going on in them?

Edited by molbio1234, 23 March 2011 - 03:18 PM.

[Adrian K]

Hi molbio1234,
Actually I will not worry too much about the quantity of the DNA being put in to run a PCR. I not sure your application, but if you really want to get precisely 1 copy to start with, I think is quite impossible. Although theoretically it works, but in practical it is not happening. If you get some journals regarding detection, you will know in order for PCR to work, it just requires more than one copy.

Refer:
http://jcm.asm.org/c...pmid=20631108   look into the section "Detection limit of multiplex nested PCR. "
http://ddr.nal.usda....33481.pdf  look for the last 5 lines of the abstract and they explained it required more than 1 copy of dna for the PCR to work
(If I do not state any references Ameya will whack me again... )

Just put your DNA into the tube and do the PCR, and you will know the quality and the quantity whether it is too much or too low yield based on the agarose gel.

I don't have those books for answer to your question. But if you can precisely tell me about your problem in your research, we can try to figure it out.
Adrian

[molbio1234]

thanks adrian. I don't have a specific research problem myself, but just trying to learn the different applications. Right now, I am interested in learning about what can cause PCR bias, and your links have been helpful. I'm sure it's probably also true number of copies of the DNA you put into the PCR reaction can influence PCR bias and I am trying to get more details about this, but most books don't talk of this, they mostly just have a generalized statement that PCR can be done with one copy of DNA and can be amplified into millions of copies after PCR. But what I'd really like to know is what happens if you have a low number of copies of DNA to put into your PCR. Or if your research was really tricky and you had to put in x number of copies of your DNA, then how to go about figuring out what x is to amplify your target correctly and avoid PCR bias. Have you heard of any books or resources that give this information?