Jayakumar, R R.Jayakumar at roswellpark.org
Thu Mar 30 16:53:09 EST 2006


Yes.. You are right.  But unfortunately, all tools only calculate the Tm
since annealing temperatures are very much dependent on the PCR machine,
purity of your DNA, buffers etc etc.. The Tm value will give you an idea
of the annealing temperature which is generally 5 degrees lower than Tm.
I am sorry that I did not elaborate on that.  
    Generally, I start my optimization PCRs (with my buddy gradient
thermocycler) by taking an annealing temperature that is 5 degrees lower
than caculated melting temperature(Tm) and work  5 degrees both ways in
my first gradient PCR.  For eg. If the Tm is 65 C, take a gradient
between 55-65. I also try out 1 - 2.5 mM Mg conc. As well as 2-3
different template concentrations.  All 3 variables can be checked on a
single PCR reaction setup in a gradient thermocycler changing only one
variable in each reaction.  If cDNA is used, use 1 ul of cDNA as
template (from 25 ul of cDNA from 1ug of RNA).  If total DNA is used,
start of with 50-100 ng of DNA.  Plasmids require very less anywhere
from 10 ng to 25 ng should be more than sufficient.  
  Best of luck
Jai

-----Original Message-----
From: vimaleswaran santhanakrishnan [mailto:vinusa15 at yahoo.com] 
Sent: Thursday, March 30, 2006 10:31 AM
To: Jayakumar, R
Subject: 

hello jayakumar,
      melting temp. cannot be annealing temperature. 
vimal



--- "Jayakumar, R" <R.Jayakumar at roswellpark.org>
wrote:

>  You can calculate it using several online tools.
> One of my favourite is
>  
ttp://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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