in the gel, DNA ladder is not resolving into bands but is seen as smear. i checked the pH of the TAE buffer and made fresh buffer but the result is same. kindly help.
- Old ladder will start to degrade and result in a smear
- Very occasionally I have seen this with older agarose
Most likely your DNA Ladder has degraded somehow (is it too old?).
Best way to go forward is to obtain new Ladder and do everything with fresh materials. Hopefully that will work and see you through for the next few months until something crashes and the whole cycle has to be repeated. Best wishes and good luck.
Hi:
in addition to the former answers , the to low voltage or to high voltage can cause this .
if this gel run for more than 1 hour . you will need to increase the voltage
@Laurence, Aditya- we have checked this by loading fresh DNA ladder but even then it was forming smear and the same ladder when it was run in another lab was resolving into bands. we have even bought fresh agarose but same result as smear.
@Hayder , Ahmed- we usually run the gel at 80V .We will try by further decreasing the voltage.
Hello,
Considering all the possibilities I would like to suggest you to change power pack and check. Power pack might having different voltage than displayed.
Hope this will help you.
@ Mangesh- hi.. we have checked that too..but no luck..same smearing..
Dear Manvendra,
Let me Know two things
1. What base pair ladder u have loaded in Gell...
2. If your gel is 1% gel or 2% gell...
Because its generally seen that 100bp (Low base pair ) ladder can not be Resolve clearly and seems to be smear in appearance in 1% GEL.
Moreover multi time use of the same Gell by remelting also gives the u said results.
If i am not wrong your are trying to load 100bp lader in 1% agarose GELL.
If it so i request you to please prepare fresh gel and Load and Hope you will get and end solution.
I will add 2 more things:
- For a 1% agarose gel 80v is perfectly reasonable. I routinely run samples on 1% agarose gels @ 120v with absolutely no smearing
- If you have changed the agarose, made fresh TAE buffer and you are using and the same aliquot of ladder is resolving well in another implying that the ladder itself is not degraded (prolonged incubation over months @ 4C instead of -20C can result in ladder degradation) then my final suggestion would be how you dissolve the ladder in TAE:
- I routinely boil for 2 min @ high power; remove from microwave; swirl; and boil for a further 1-2 min to completely dissolve agarose grains: Incomplete dissolving of agarose can cause resolution problem, so tends to be exacerbated in 2-3% gels where you require at least 3 minutes of boiling for standard microwaves
I agree with the procedure of Mr. Laurence Stuart Hall and would like to add one more line that add Ethidium bromide when it cools a bit and avoid adding at high temperature.
Hope it resolve the trouble.
@Srinivas- hi! we dont reuse the gel..we make fresh gels..yes this gel was 1%..and the ladder was 100bp. i will make fresh buffers and then make 2% gel..
@Laurence- yes i have changed agarose so i will make fresh TAE buffer.. thank u for ur suggestion :)
hi,
I suggest you to dilute your ladder and using as (1ul lader+1ul loading die +3ul TBE or TAE) then add your ladder 5ul to the well it will be good for using ladder even your ladder is ready to use and having loading die in it.
We had results like this when we were trying new "safe" ethidium bromide alternatives. Especially Diamond from promega was prone to artifacts (we dissolve it into the gel prior to electrophoresis). Besides, it seemed to be very unstable compared to other dyes (in post-stain protocols, you should be able to reuse the buffer 2 or 3 times; in our hands the second usage was very poor compared to the "fresh" solution).
To solve the electrophoresis aberrant results we used post-stain protocols and afterwards changed to Gel Red from Biotium.
as your samples are a good distance from the wells it looks like the gel has been run too high a voltage and the smearing is due to heating of the gel and running too fast. Check it with a different TAE and power supply in case one of them is misbehaving. The ladder really has run a long way. Lower voltage is also worth a try and check that the current is as usual for your gels
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