9 replies to this topic

[shamim]

Hi
I had to design primers having the desired restriction sites at 5' of both; the reverse and Forward primers and I was left with the option of primers having different annealing temperatures (Forward, Ta 61 and Reverse Ta 57).  PCR by using these primers is giving smearing, I tried different annealing temperatures.  I can not redesign primers and these are the only sites I can use. Any suggestion, much more appreciated.  Thanks
Shams

[nabla]

Have you tried different Mg-concentrations(e.g. 1-4mM)? Perhaps you should reduce the annealing time, but this depends on your amplicon size. You can even try an asymmetric PCR with a different amount of one of the primers - , after you have checked if reducing the primer concentration in general has an positive effect on the reaction- .

[shamim]

Hi nabla
Thanks for your advice, have tried different annealing temperatues but did not try different Mg concentration.  will try that.

[shamim]

Hi
I did try different Mg concentrations and also did asymmetric PCR (varying concentrations of the R and F primers) but still no success, got smearing, no bands.  Any other suggestions please.

Thanks
shamim

[nabla]

shamim, on Dec 14 2004, 09:23 PM, said:

Hi
I did try different Mg concentrations and also did asymmetric PCR (varying concentrations of the R and F primers) but still no success, got smearing, no bands.  Any other suggestions please.

Thanks
shamim

What about hot start pcr. How long is you amplicon and what is your target: genomic, plasmid? Is your traget pure enaugh? More infos please?!!

[shamim]

Hi Nabla
I always do hot start, target is plasmid and its been purified by Qiagen columns, the amplicon is 476 bps.  After using PCR enhancer, I did get some bands in the area spanning 200 - 500 bps.

Regards

Shamim

[nabla]

Perhaps you should extend the elongation time a bit, cut the plasmid or use a kit for difficult amplicons (GC rich). The last chance is to make a nested pcr?!
Perhaps it is a steric problem and the Taq is kicked off the target?!!
... I have no further tips, sorry.

[rajgene]

will this help u any way.

http://www.stanford....R_reactions.pdf.
have u calculated the annealing temp for the first cycle without the rest.sites and xtrabases? may be this works... tell me if it does work.
rajgene

[Gouwetonny]

Maybe the use of a Touch Down PCR wil help

[luminb]

what is your template source for pcr? RT, genomic DNA, or plasmid? tm around 60 is kinda low. especially bad for RT product and genomic dna. you better redesign the primers. longer primers will raise up tm and make pcr more specific. if you can't, then try to add some co-solvent in the solution. dmso or glycerol. check some webpages from manufacturers of dna polymerase.  they will give you some good troubleshooting.