14 replies to this topic

[minty]

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

[perneseblue]

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

[T C]

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

[mastermi]

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...

[minty]

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

[microgirl]

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

[perneseblue]

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.

[minty]

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........

[ah6tyfour]

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/n...roductF-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 =)

[minty]

ah6tyfour, on May 6 2009, 09:06 AM, said:

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 =)

ya i had tried a range from 56 to 62 but i didnt got any ampilfiacation at any mgcl2 conc......
so i shifted to further low annealing temp.........but at 50 i got smear ..........nw wat ????

[ah6tyfour]

Did you design your primers using some sort of program like Primer3?  If you designed them by hand, you might want to make sure you are ordering the correct thing.  I've had many a student spend a month on a troublesome PCR only to realize there was a mistake in the sequence of the antisense primer.

Also, did you BLAT your primers with Genome Browser to make sure they're specific?  Also use Genome Browser's PCR function so you can paste in your sense and antisense primers and have it predict the product.  Make sure the product is the right size and in the right region of the genome.

And what is your template?  Are you sure it's good?  I've had PCRs fail because the DNA went out of solution so I ended up diluting it way too much (like gDNA at 600ng/uL until it all spindled together).  What liquid is your template in and what volume of template are you adding?  If your DNA is in TE and you are adding a lot of it, you might inhibit the reaction.  Same with cDNA from an RT reaction.  Add more than 10% of your PCR volume and the salt from the cDNA will start messing up your PCR.

You can also try Touchdown PCR.  The annealing temp will increase a little bit each cycle for the first 10 cycles or so....and the hope is that you'll get enough amplified at some temp that it can start acting as good template.

If everything checks out and you are getting nothing, use Phusion Polymerase....you will get it.

Edited by ah6tyfour, 07 May 2009 - 12:30 AM.

[minty]

ah6tyfour, on May 7 2009, 12:25 AM, said:

Did you design your primers using some sort of program like Primer3?  If you designed them by hand, you might want to make sure you are ordering the correct thing.  I've had many a student spend a month on a troublesome PCR only to realize there was a mistake in the sequence of the antisense primer.

Also, did you BLAT your primers with Genome Browser to make sure they're specific?  Also use Genome Browser's PCR function so you can paste in your sense and antisense primers and have it predict the product.  Make sure the product is the right size and in the right region of the genome.

And what is your template?  Are you sure it's good?  I've had PCRs fail because the DNA went out of solution so I ended up diluting it way too much (like gDNA at 600ng/uL until it all spindled together).  What liquid is your template in and what volume of template are you adding?  If your DNA is in TE and you are adding a lot of it, you might inhibit the reaction.  Same with cDNA from an RT reaction.  Add more than 10% of your PCR volume and the salt from the cDNA will start messing up your PCR.

You can also try Touchdown PCR.  The annealing temp will increase a little bit each cycle for the first 10 cycles or so....and the hope is that you'll get enough amplified at some temp that it can start acting as good template.

If everything checks out and you are getting nothing, use Phusion Polymerase....you will get it.

yah i had designed my primers by primer3 only....
and yes i had checked their specificity too and both sense and antiensse match well.......
m using genomic DNA as my template and it is in TE ....

[swanny]

Redesign and resynthesise. 2(A+T) + 4(G+C).

[minty]

swanny, on May 11 2009, 12:23 AM, said:

Redesign and resynthesise. 2(A+T) + 4(G+C).

thanks a lot 4 advise

Edited by minty, 11 May 2009 - 12:46 AM.