PCR triplicates versus one reaction - differences? (Mar/21/2011 )

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

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.com.my/books?id=vGvQkK1tAx8C&printsec=frontcover&dq=PCR+Troubleshooting:+The+Essential+Guide&source=bl&ots=zdc1-hJoZj&sig=KmiJAgLu5TWXs-QJ0QofH5HZRdQ&hl=en&ei=vtGJTa-RJceGrAf_6rziDg&sa=X&oi=book_result&ct=result&resnum=9&ved=0CEwQ6AEwCA#v=onepage&q&f=false

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

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?

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/cgi/content/full/48/9/3165?view=long&pmid=20631108 look into the section "Detection limit of multiplex nested PCR. "
http://ddr.nal.usda.gov/bitstream/10113/25690/1/IND44133481.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

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?

I am getting the feeling my question in the above post is more of a trial and error, which is a pain . Does anyone have any guidance or suggestions ?

No reference this time.

If you know your gene sequence, design a primer. If you can extract and purified your DNA, make it in 10pg concentration. Do a 10x dilutions till 1fg level.
Run your PCR in gradient.

You have to do all the above (unless I missed out something...) in order to answer your question. You will know the minimum template DNA required for that particular PCR to work. No general or common or fixed number of amount.

I not sure what you mean by PCR bias. Bad primer design: unspecific amplification.

too low maybe no amplification

molbio1234 on Wed Mar 23 03:07:23 2011 said:

If you put 50ng in 50ul, you have a concentration of 1ng/ul. If you put 50ng in 100ul, you have a concentration of 0.5ng/ul but you still have 50ng in your tube.

As for the low copy number PCR, we usually increase the number of cycles in order to detect PCR product on a gel.