Anyone use the Sigma product ReadyMix Taq PCR Reaction Mix with MgCl2? Having problems with my PCR products, essentially I have no amplification, and only primer dimer. Just wondering if anyone has experience with this specific product...I typically do a 25ul reaction however I may try the 50ul as per the Product information sheet (information sheet does say you can scale down the reaction size...)
This could be for a number of reasons that you may need to look at in a bit more detail:
- Annealing temperature
Are you using the correct temperature for your primers (e.g. 60degC for primers of 65degC Tm)?
- DNA template concentration
Too low a concentration may result in lack of amplification, and possible primer dimer
- Concentration of MgCl2
Can affect efficiency and overall amplification
- Contaminants reducing taq efficiency, or primer annealing
Scaling down shouldn't make much of a difference, unless it is a DNA template concentration issue - thus, using half as much means less likely to amplify if the concentration is low to begin with. You can adjust the water and DNA template ratio to give you a higher starting conc. of template.
Hi Daniel, thanks for the tips! I'll have to give these a go. It just recently became an issue when we switched to a different ReadyMix (which tells me that's the problem) but I am still going to try the things you mentioned just to rule it all out. Thanks!
- 25ul for a ready reaction should be perfectly OK
- You shouldn't need to scale up to 50ul
- I agree with the previous speaker that you might have to optimise your PCR
- In addition if you are not already doing so try commencing your PCR with 1 denaturing cycle of 96C for 5 min and also increase the cycle number by 5, e.g 35 from 30
- In addition if primer dimers are an issue add 0.5 M Betaine to your PCR reaction or alternatively 5% DMSO:
- http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011024
- http://bitesizebio.com/19420/just-what-do-all-these-additives-do/
- Finally to obtain more consistent results in addition to using additives and modifying your PCR as suggested you could invest in better designed primers with a lower propensity for forming hetero (primer) dimers. I find the IDT primer design freeware particularly useful in that regard
- https://www.idtdna.com/calc/analyzer
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