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Conventional PCR problems - wisamni2004 (Nov/16/2010 )

Hi , i hope some body can help me

1st- if i am doing a PCR and the amplicon is less than 100 bp how can i differentiate it from primer dimers .

2nd - how much is the least DNA concentration i could work on with PCR , and if it does appear in the gel does that mean it is of good quantity .

3rd - if i am using a published protocol for genotyping certain genes and i am using the same primers should i optimize temperature and Mgcl or i just stick to the same protocol.

thank you

-dr.wisam-

Hi,

to answer briefly:
1. primer dimers are usually smaller than 100 bp and they can be seen using a higher concentration gel - 2% or more. In the image I attached you can clearly see the difference between primer dimers and the amplicons. Also if the amplicon is being effectively amplified (left side of my image) the amount of DNA is much larger than the amount from primer dimers. The latter usually give a more fuzzy diffuse band (right side).
2. Theoretically one copy of DNA is enough for PCR, but practically any amount that you have will do (as long as it is not too much). Just set the number of cycles to 35 and you should amplify any small amount of your template. Sometimes (and believe me this is a real pain in the a**) you amplify your product even if it is not supposed to be in the tube (e.g. negative control sample).
3. Yes. The buffers from various suppliers contain various amounts of Mg and other ions, making any standardization wishful thinking. However, this is more a problem for qPCR where you quantify the product as it is being amplified. In conventional PCR you mainly want your product for subsequent manipulation, and PCR is a very robust method, so even if the precise conditions are not the same, the product will still be amplified.

Perhaps other PCR gurus will add their thoughts...
Cheers,
Miha
Attached Image

-BioMiha-

BioMiha on Tue Nov 16 11:49:22 2010 said:


Hi,

to answer briefly:
1. primer dimers are usually smaller than 100 bp and they can be seen using a higher concentration gel - 2% or more. In the image I attached you can clearly see the difference between primer dimers and the amplicons. Also if the amplicon is being effectively amplified (left side of my image) the amount of DNA is much larger than the amount from primer dimers. The latter usually give a more fuzzy diffuse band (right side).
2. Theoretically one copy of DNA is enough for PCR, but practically any amount that you have will do (as long as it is not too much). Just set the number of cycles to 35 and you should amplify any small amount of your template. Sometimes (and believe me this is a real pain in the a**) you amplify your product even if it is not supposed to be in the tube (e.g. negative control sample).
3. Yes. The buffers from various suppliers contain various amounts of Mg and other ions, making any standardization wishful thinking. However, this is more a problem for qPCR where you quantify the product as it is being amplified. In conventional PCR you mainly want your product for subsequent manipulation, and PCR is a very robust method, so even if the precise conditions are not the same, the product will still be amplified.

Perhaps other PCR gurus will add their thoughts...
Cheers,
Miha


1. I agree with above. You should be able to see primer dimers on a 2% gel. My amplicon are 50-100bp and i can always distinguish between primer dimers and my target. I'd also suggest running a melting curve program after your PCR program. If you have only 1 distinct peak its highly unlikely that primer dimers are forming.
2. The amount of DNA neccessary really depends on a number of factors. Its all about your experimental setup and how efficient your PCR is.
3. Also agree with above. I'd start with the reference protocol and then use that to optimize for your own experiments. Not only will reaction mixture formulation affect your primer specificity and amplification efficieny, but PCR program (ie. duration, 2step vs. 3 step, annealing temperature, PCR machine used)

-biotechgirl-