I am running a Multiplex, and I would like to know if someone has some good idea how to increase my gel quality. So I am using 3 primer set and different DNA. I always integrate my Water control as last (the one to the right, before the marker) and before there is a blank space, to see if there is any well jump from one to another pipetting. I see the bands quite well, but I have this "smear" around the band and I don't know how to get rid of it. For a PCR reaction I normally load 12.5 microL on the gel and let it run at 85-95 V (2% agarose gel, 200 mL). I use the following MasterMix (for 9 samples + 10% pipetting mistakes):
Buffer 40 (microL)
dNTPs 4
MgCl 8
Primers 5
(Dream)Taq 1
DNA 1
up to 200
For the concentration I use to dilute my primer stock 1:10 and keep it separately from the stock itself.
For the dNTPs I use d(A/G/C/T)TPs 2.5 mM
the rest is pretty standard and is used from the DreamTaq kit.
Is it possible that my MgCl/dNTPs ratio is not optimized?
I think this might be my problem, because my DNA is quite ok (quality is acceptable, around 1.6-1.8).
EDIT: sizes in different samples are different because of different DNA has been used. I am not interested in that right now, just to increase my bands quality and to avoid primer dimers/degraded DNA (lower bands)
Thanks in advance!
Best
Hi Francesco,
This problem is probably caused by melting temperature of the primers (Tm), template or MgCl2 concentrations. When you got a single band, you can get a stronger band by higher number of cycles. Also, you can firstly heat the primers and water mix to 98 degree then you can add other compenents.
Good luck
You could try using less primer. Also a hot start either with a hot start enzyme or manual hot start as Aydın Yeşilyurt suggests. With less primer dimer you can expect better amplification. Also run the gel slowly because fast gels are hot and bands will broaden by thermal diffusion.
You can change the primers some time it happens with me also but when i change the primers then the result is okay.
Thanks to everyone for the suggestions.
For the MgCl2 concentration, would you suggest to increase or decrease the ration between MgCl2 and dNTPs?
I am trying as well with a decrease of primer concentration (1:10) but from previous experiment I think that the ratio between amplified DNA and primer dimers is still high so I will experience still primer dimers with diluted primers.
If anyone has some other suggestion, please bring it! I appreciate your effort.
Best regards.
well, try:
1) different concentrations of MgCl2 from 0.5 to 5 mM.
2) different annealing.
3) different additive (alone or different mixtures):
dimethylsulfoxide (DMSO; at a final concentration of 1-10%),
formamide (at a final concentration of 1.25-10%),
bovine serum albumin (at a final concentration of 10-100 μg/ml),
Betaine (at a final concentration of 0.5 M to 2.5 M). However attached a very useful article
Probably I should have added before:
https://www.thermofisher.com/order/catalog/product/B65
The DreamTaq already contains MgCl (20mM) however I thought that increasing the concentration of MgCl2 would have increased the quality of my bands. Is there any paper (even a technical review) which states the limits of concentration for MgCl2 or dNTPs?
In the article suggested by dr Raniero Iraci (thanks for the sharing) it is possible to observe that the optimal concentration of MgCl2 is indeed around 2.0mM, but is it possible to say more about the relationship between reagents, such as dNTPs/MgCl2 ratio?
Thanks again for your feedbacks.
Best regards
Well, I recommend to do empiric tests with both the MgCl2 and the various additives. Much depends on the DNA and the combination of reagents and additives. There are no universal conditions
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