For a Mg2+ titration, what concentrations are best to pick? I mean, exactly what values should I be trying?

Mg2+ at a final concentration of 1.5, 2.0, 2.5 and 3.0 mM should be tried.

I'm trying to make more efficient an RT-PCR because, albeit i have my desired PCR product, i wish to get a better reaction. I think the problem is that the primers are getting in dimers. I've heard about the use of detergents or DMSO in the reaction mix but i'm not sure about this. Does anyone knows a way to do it without change the Tm?

Aleruiz

Dimers tend to form when the template concentration is low. If you can, try titrating in more of the template.

Adding yeast tRNA carrier can reduce the amount of template that sticks to the tube walls, making more of it available for amplification.

When designing your primers, check them for homo-dimer and hetero-dimer formation with the Oligo Analyzer program at the IDT website www.idtdna.com.

dmso can be used between 2-10%, but the higher you go the less active the polymerase becomes so you may no get the same amount of cylces. u can try at 2 and 5% to be safe.

100bp sounds too big for primer dimer - your oligos would need to be >50 bases each to get that - looks like a proper pcr product. MgCl2 titration might help, as well as trying different Tms.

1) Try the DMSO up to 5%. Some polymerases are ok with DMSO and others aren't. You never know until you try.

2) Try a two step PCR. There is a paper in Biotechniques about a two step PCR reaction but I don't have the reference with me since I'm at home and it's in lab. Its worth looking up. Basically you've got template plus forward primer in one tube and template plus reverse primer in another tube and then do between 1-15 rounds of PCR. Then combine the contents of the two tubes and do your normal 25 rounds of PCR. Sometimes this allows the primer to bind to the template effectively and allow you to get some initial transcripts which will then bind to each other when you combine the contents of the two tubes.

3) If you're not using a polymerase for high GC content, then get one. It will save you a lot of hassle. It won't solve your problem but it will help you narrow down things when troubleshooting. KOD XL polymerase (novagen) isn't sensitive to DMSO up to 5% and it helped me get all of my mutants but not without a lot of tries just because the GC content of primer and template were over 60%. Some people like the quik change kit by stratagene but I never got it to work, which is really expensive when its $600 a kit for 30 rxn or something like that. To each his own.

4) I wouldn't go below 55 deg for an annealing temp. 50 might be fine but below that you're pushing your luck with trying to have a high enough temp to resolve any hairpin structures which are common in high GC rich primers not to mention possibly decreasing the specificity of matching primer and template.

good luck! it takes a lot of troubleshooting and its hard to get consistent results.

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bouttela on Mar 17 2003, 08:01 AM said:

i design primer but Gene not identification until now from mycology but identified with higher plant, designed primer must the band of gene appeared at 420 BP as higher plant but appeared sharp band at 150-100 BP only and primer-dimer not formed for control or may pale pale band and disappeared with time, this may be specific band or not.

saly
saly4ever@hotmail.com

do you have try tochdown pcr