How to get rid of PCR primer dimer - (Mar/17/2003 )
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?
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.
I have designed primers with restriction enzyme sites at the 5' and 3' ends respectively and am having trouble getting the desired product. Template is the gene itself gel purified and keep getting bands at approx 100 bp. I'm thinking that these are primer dimers...Don't want to have to redesign primers. Another problem is the Tms are high - 68 .
New frustrated tech
May be you can try by reducing your primer concentrations.
It might be as simple as using a Hot-Start polymerase. I think that Vent is for higher fidelity, whereas hot-starts help to remove any impurities or non-specific bindings, dimers, etc. We had dimers in our gels, and switched to a hot-start called RepliTaq Thermal and it did the trick.
Try reducing your primer concentration. What is it by the way.