Hello guys,
I have performed experiment on optimisation of PCR for one of the SNPs and while performing electrophoresis, the issues occurred that I'm trying to figure out right now, and need some help with that.
The methods followed while preparation of the gel:
In order to prepare agarose gel, first preparation of 100ml of 10X Tris-Borate-EDTA (TBE) was needed. To obtain that, 1.080g of Tris Base, 0.550 of Boric Acid and 0.095g of EDTA Na2·2H2O was added to the beaker. It was then filled with reverse osmosis (RO) water, after which HCl has been added to adjust the pH of the substance to 8. Substance was stirred well while HCl and RO water has been added to achieve final volume of 100ml of the TBE. Four new, clean beakers were taken, to prepare two 2% agarose gels with 12 wells, which had the best porosity for the size of amplicon that was studied in this experiment. 5ml of TBE has been pipetted carefully into each one of them, and then diluted with 45ml of RO water to achieve 1X TBE buffer. Then carefully measured 1g of agarose has been added to two of the beakers, and 2ul of GelRed, before microwaving them for 2 minutes to dissolve all particles, after which they were poured into the gel tanks, and left to set for around 20 minutes, before removing end plates and combs. TBE buffer from the other beakers has been poured all over set gels.
4ul of loading dye has been added to each PCR tube and briefly vortexed. 5 ul of DNA ladder has been loaded into first well of each gel. Then one gel was filled in order with 12ul of PCR samples of set A, the other one with duplicate set B. Loaded gels then had been run at highest current for approximately an hour, until the samples has travelled at least halfway through the gel, after which photos of the gel image has been taken.
The problem already was obvious once the run was over, visible smiley blue line on top of the gel, which I assumed was either the issue with the pH of the TBE (however as I had TBE that I used and checked it for confirmation, it was still at perfectly 8). Another thing is that the blue line has decolorized in few spots, into greenish/grayish colour which I cannot find any explanation for in troubleshooting pages.
Then once trying to obtain gel images, it turned out gels were melted, even though the tanks were not warm at all when I took them to do the images, which was straight after turning the power pack off.
The images don't show any good results, they are having the burn line, DNA ladder didn't separated itself, there are some faint bands visible in one gel, there are visible DNA bands in the wells, so some DNA didn't even left the well.
Did something similar happen to anyone and what is the main issue starting with for this experiment?
Thank you so much in advance for any response.
You said you used the "highest current". That's your problem right there.
Most folks don't go over 120 Volts.
You melted your gels by literally running them too hot.
You do not need to titrate TBE with HCl to adjust the pH. By doing that, you introduce fast moving Cl- ions, which might be responsible for heating the gel and distorting the bands. Just mix the specified amounts of Tris, Boric acid, and EDTA, and add water to them to the final volume. Without any adjustment, the pH value should be within 8.3-8.6, which is perfectly fine for nucleic acid electrophoresis.
If due to some reason you need to set the pH precisely to 8.00, add more Boric acid to the above mixture while stirring it.
Also, you may let the gel to sit for about 1 hour at RT after casting to make sure it is properly solidified. And I would rather add the GelRed after microwaving the agarose and cooling it down to ~ 50 oC rather than before.
Green color of Bromophenol Blue dye points to the local pH drop, which is likely caused by acidification of the anodic buffer due to high electrolysis rate at the anode. You need to decrease the voltage/current to remain within 80-120 V and 10-30 mA ranges.
Answers already provided by other colleagues.
"However, voltages that are too high can possibly melt the gel or cause smearing or distortion of DNA bands." (ThermoFisher Scientific)
use fresh buffer for preparation of gel as well as for filling up the electrophoresis chamber. Reduce the voltage up to 75V-80V
the voltage you used is too high, do not go over 120 Volts, it will be too hot and then the gel will melt. so go with 90 , more better
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