Hi, you said that you saw the band, the possibilities were:

came out the gel (did you run a marker near the DNA?).

overheating during the running (did you make a small or big gel? Because with small one at 85 V the band can faint for overheating).

long UV exposure when you saw the band.

Finally I would check also your reagent, make a new 0,8% gel with fresh buffer and cleaned apparatus.

Hello, since you did not find any band it means that you made an inappropriate choice of your gel concentration. 0.8% seem to be a very low concentration for DNA. I suggest 1.5% to be sure of detecting your DNA. if you still don't find it you can raise your gel concentration even more. Good luck 

is it total DNA? or a PCR product? i use 1%agarose and i resolve a ladder from 100 to 1000 bp in about 60-70 min with 100V.

I think 45 min it's too much, specially because of your gel concentration. I would make an 1% agarose gel and run the electrophoresis for 30 min with 100 V.  If it's crude DNA, you'll need a ladder with bands bigger than 5000 bp.

Hi, you said that you saw the band, the possibilities were:

came out the gel (did you run a marker near the DNA?).

overheating during the running (did you make a small or big gel? Because with small one at 85 V the band can faint for overheating).

long UV exposure when you saw the band.

Finally I would check also your reagent, make a new 0,8% gel with fresh buffer and cleaned apparatus.

I agree with Valeria that as you saw the dna earlier the probable reason is that on viewing under uv light the EtBr was quenched and possibly has broken up the dna so it is now spread over a wide size range so that at any position on the gel there is very little dna so it is not visible. You could try re  staining the gel in aqueous Etbr and see if the dna re appears or just run another gel and be quick about your viewing of the gel. If the dna remains intact it will be very high molecular weight and will take a very long time running through the gel. I think 0.8% is a good concentration for large dna but cab be a bit slippery to handle

I agree with Paul and Valeria, if this is genomic DNA, you can run on 1% gel at 100V, I usually run for an hour and I can visualize well. also make sure that u add 1 microliter of ethidium bromide to your gel, which intercalates to your DNA allowing you to see it. It ia also very important to include a ladder just so you can estimate the concentration of your DNA. you can either visualize with ethidium bromide or gel red ( in our lab, we mix about half a microliter of gel red with 1 ml of loading dye and just add 1 micro liter to every 5 micro liter of DNA sample).

Good luck

Hello Dileep, your agarose gel concentration is certainly fine for genomic DNA, people forget that 0.8% (even 0.7%) is regularly used for Southern blotting, where average DNA fragments length is only 10-20 kb. So, your problem is probably the run.

The bigger the DNA, the slower you have to run it to retain a compact band. Your Volt indication, as Valeria pointed out, is very relative, since depending on the size of your chamber, the size of the gel and therefore the resistance of the system, it could be fine or it could be too much... I would aim at 20-40 mA instead. I guess you probably also loaded quite a small amount of gDNA, so with the smearing you lost it completely, but I'm sure if you just slow down the run you'll have your gDNA.

Good luck.

The genomic DNA can be loaded on 0.8% gel so that it can move and appear as a band. In higher percentages  (2%) of agarose the DNA will stay in the well you loaded it on.

 You ran the gel for long which may have lead the DNA to run away out of gel  or sometimes the EtBr decays and the bands are faint or they just disappear and you are not able to see the band although they are there in the gel. To address this issue you can do what Paul suggested i.e. stain your gel in  Etbr solution again. 

Dear Dileep,

Apart from all the excellent suggestions given, you might want to follow these:

1. Decide about the percentage of agarose gels depending on your DNA sample, which you want to visualize and the bands you want to resolve. I always run 0.8% gels for my 1.1Kb DNA. Also for plasmids (vectors around 5Kb) I use the same.

2. Depending how you want to resolve your DNA, choose the length of the gel. I use a longer gel to see my PCR product as, it helps to resolve the 1Kb ladder resolve completely and I can easily determine the desired band without any ambiguity.

3. Also these factors decide the time of your run. If its a shorter gel, you might not want to run it for a longer time in case your fragment is big enough.

4. Running voltage should again be determined by your sample size and gel size. But I have run ~1Kb fragment at 70-80V in a long 0.8% gel for 2-3 hours for good resolution.

5. Also use optimum concentration of EtBr so that your bands do not appear faint. You can also try to stain gel after running, rather than adding EtBr to the gel itself.

Considering all these factors you should cast and use agarose gels.

Hope it helps!

Hi 

The agarose cincentration is ok for genomic DNA but the time could be long if you are running smal gel as the EthBr migrate to from positive to negative and this could lead to the presrnce of the DNA in a position not stained. I sugest you to restain the gel if this happen again.

Hello Dileep

Have you check your gel after few minutes to know DNA is moving forward  in gel?? Now  I wanna know, you have loaded genomic DNA or any apmlified gene? If you loaded Genomic DNA then it should not overflow, if it occur, there is problem of DNA conc..If amplified gene then you have to increase percentage of agarose..

DNA could be run in 1% gel in 80V, 45 min 

Hi Dileep,

My thoughts are;

the lesser the concentration of the gel the faster the migration will be. This is a factor of the matrix created by the gel molecules. Perhaps use a much higher gel concentration. If you still wont see the band then you might want to recheck the DNA concentration that you obtain from the extraction protocol you are using.

Hi Dilip,

 The provability of DNA gone is yes. Did you check the gel with 45min? did you saw any band over the gel? if you didn't check within 45min then the possibility is high that the DNA gone. Because as much as thin gel you will use that much fast the DNA will run through the. And you have to also consider the voltage, in high voltage the DNA also run over through the gel.

Hope it will helpful for you 

Preferably, run 1% Agarose gel with 100V for at least 50 minutes to be safe. I had experience with lower concentrations of agarose and the bands were not too visible so had to retry with 1% and was very good.

Hi, This depends on the fragment you need it and what about the DNA marker?. I think 70 V for 45 min. enough for check your gel.

Best regard

I always made 0.8% gel for genomic DNA and run gel at 85 volt ,never found this kind of issue.please check you buffer 

Hi

I am agree with Valeria De Arcangelis, you shoud laod marker and check your gel after 10 min of running.