# Determine annealing temperature of primers - (Apr/26/2009 )

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hey can any one plz help me to decide annealing temperature for a set of primers with tm 59.4 and 66.4

-minty-

minty on Apr 26 2009, 03:09 AM said:

hey can any one plz help me to decide annealing temperature for a set of primers with tm 59.4 and 66.4

When faced with primers that have a large difference in tm, calculate the melting temperature using the primer with the lower tm.

Try 55 C and work from there.

However should this PCR fails to work despite your best effort, you should consider redesigning the tm 66C primer and aim for a tm closer to 59C

-perneseblue-

Yeah........55 should work. I don't think you would need to redesign but if you do you can either add one codon in the 59 primer or take out one from the 66 to adjust the Tm.

Best,
TC

perneseblue on Apr 26 2009, 05:04 PM said:

Try 55 C and work from there.

However should this PCR fails to work despite your best effort, you should consider redesigning the tm 66C primer and aim for a tm closer to 59C

-T C-

If you have the possibilty to use a gradient cycler, just try out temperatures between 55 and 65 degrees in two-degree-steps to see what is working best...

-mastermi-

mastermi on Apr 26 2009, 01:24 PM said:

If you have the possibilty to use a gradient cycler, just try out temperatures between 55 and 65 degrees in two-degree-steps to see what is working best...

i have tried a range of 55 to 65
i got three unspecific bands at 55 with 2.5 mM MgCl2
than 3 different unspecific bands at 58.5 with three conc of MgCl2 i.e. 1.5 mM , 2.0mM, and 2.5mM
and no furtherresults on any temp up to 65
nw wat should i do ???????????
my required product size is 1.7 Kb

-minty-

Sometimes primers just don't work well together. Can you redesign and try new ones?

-microgirl-

hmm... are you certain that there is no hint of your desired PCR product?

What type of template DNA are you using? If it is genomic DNA you could try diluting some of the DNA with sdwater (1:20 to 1:50 dilution) and use that diluted DNA as the template.

Sometimes despite your best effort at clean it up, the genomic DNA remain contaminated with PCR inhibitors. One way around this is to dilute out the inhibitors by diluting the DNA. PCR will then be able to amplify the DNA to a detectable concentration.

If you still can't get some indication that that PCR is working you should consider a new primer.

-perneseblue-

perneseblue on Apr 30 2009, 09:20 PM said:

hmm... are you certain that there is no hint of your desired PCR product?

What type of template DNA are you using? If it is genomic DNA you could try diluting some of the DNA with sdwater (1:20 to 1:50 dilution) and use that diluted DNA as the template.

Sometimes despite your best effort at clean it up, the genomic DNA remain contaminated with PCR inhibitors. One way around this is to dilute out the inhibitors by diluting the DNA. PCR will then be able to amplify the DNA to a detectable concentration.

If you still can't get some indication that that PCR is working you should consider a new primer.

ok i ll try out for this problem ..............i have one another set of primers for separate region of gene
primers have tm 61.9 and 67.2 ........
with these am getting smear in my pcr at annealing 50 ..........
any gudie line plz........

-minty-

If the area you want to amplify forces you to design primers that have melting temps that different, you might want to try the Phusion polymerase (Finnzymes...sold by NEB now). Use the Finnzymes website primer Tm calculator and then use an annealing temperature 3-degrees ABOVE the melting temp of your lower primer.

http://www.neb.com/nebecomm/products/productF-540.asp

I keep this around the lab for use with difficult PCR and it usually doesn't let me down.

You only need 1U of the enzyme per 50uL reaction so the smaller \$125 kit is good for at least 100 reactions. I usually do my reactions in 20uL so one tube of this Phusion enzyme lasts me 300-400 samples. Make sure you read the instructions though...temp settings and times are very different (15 seconds per kB extension time). A specialized buffer for amplifying GC-rich regions is also included with the enzyme.

-ah6tyfour-

As for your second set of primers....you should always start with annealing temp = Tm - 5 degrees. So if your lower primer is 61.9, you should start with 58 (although for anything around 58 degrees to 62 degrees I usually use a 55-degree anneal because it's the default of my saved protocol). But 50-degree annealing for a 62-degree Tm primer is very low and you're not going to get very specific annealing.

Phusion will take care of both =)

-ah6tyfour-
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