Annealing Temperatures for PCR

Deanne Bell dbell at fresno.ars.usda.gov
Thu Mar 30 11:01:37 EST 2006


I totally agree with Jai - you really need to run your sequences thru a
calculator to get a GENERAL idea and then run PCRs with a range of
annealing tempuratures (I LOVE my gradient thermocycler).  Then you will
want to optimize the amount of Mg++ and the amount of DNA added for your
specific type of samples.

  
For example:
* plant DNA seems to prefer ~4mM MgCl, while the bacteria I work with
amplifies best with 1.5mM. 
* some DNA extraction protocols will leave left-over substances that are
inhibitory to PCR thus less of the DNA prep must be used to get good
amplification (this is really counter-intuitive when you see light bands
being produced).  Too much DNA will throw off the PCR reaction also.

Good luck, hope this helps
Deanne Bell

 
 
 

>-----Original Message-----
>From: methods-bounces at oat.bio.indiana.edu 
>[mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of Jayakumar, R
>Sent: Thursday, March 30, 2006 6:35 AM
>To: methods at magpie.bio.indiana.edu
>Subject: RE: Annealing Temperatures for PCR
>
> You can calculate it using several online tools. One of my 
>favourite is
>   http://www.basic.northwestern.edu/biotools/oligocalc.html
>You can also search on google with keywords "annealing 
>temperature calculator tool".  The melting temperature is your 
>annealing temperature.
>
>   But still the actual Annealing temperature has tto be 
>optimized through gradient PCR using different conc. Off Mg 
>and salts.  The Tm values only helps to give an idea.
>Best of luck
>Jayakumar
>
>-----Original Message-----
>From: methods-bounces at oat.bio.indiana.edu
>[mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of 
>retnohh at mail.mitra.net.id
>Sent: Wednesday, March 29, 2006 6:31 PM
>To: methods at magpie.bio.indiana.edu
>Subject: Annealing Temperatures for PCR
>
>Dear sir,
> From internet I know that youa an expert in Molecular Biology 
>research.
>I have a problem with my student PCR.
>If you do not mind, please help me to tell me how to calculate 
>the exact annealing primers for pair of primers:
>5'- TTA GGG CAA GAG ATG GTA AGG -3'  and
>5'- TTA TAA CAA TGA TGG AGG G -3'
>Thank you very much for your kindness.
>
>Best regard
>Retnohh
>
>
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