There are two ladders (1kb plus) at 1st and 9th lane. Amount of the ladder loaded was 4ul. 2nd and 3rd lane are undigested and double digested samples respectively. lane 4,5 and 6,7 follows the same pattern. Undigested sample - 3ul double digested - 2ul. voltage- 100 for 1st 40 minutes, 120 for next 20 minutes nd 150 for 20 minutes, as i was running out of time so i had to increase the voltage. As per my knowledge, it does not has anything to do with the voltage so what could be affecting it?
If you increase the voltage due to the increasing resistance the gel will heat up and of course this influences the bands, in the worst case the gel will partly melt.
Try a gel with less agarose and less sample volumen.
Hi Ishwar it looks like too much DNA was loaded to me (1-2 ug of DNA should be sufficient for visualisation with Ethidium).
Another reasons for aberrant running in a gel is that the buffer (TAE/TBE) contains to much or too little salt/electrolytes (i.e. not properly made up - I have done this by accident).
Too much and the DNA does not really leave the well (as all the current is being carried by the electrolytes in the salt) too little and the DNA will "streak" though the gel as this is the only thing present capable of carrying the current apart from the water.
A third reason for "streaking" in the gel is the voltage is too high for the % gel. The voltages sound about right but it depends on the buffer type (TBE/TAE) as well as the gel size. If you are running a LMP gel or a low % agarose gel drop the voltage compared to a normal (i.e. 1%) agarose gel (as they have a "looser" matrix for the DNA to move through). One indication for this is that you will get a different current than normal at a fixed voltage - a lot of people will run a gel at a constant current rather than a fixed voltage (as current drops over time due to increased resistance as the gel heats up).
Sorry I can't be more help - personally I would a) re-make the buffer b) take one of the digestions and run various amounts or c) run at a lower voltage for a longer time.
Gary
Answering following questions may help you to troubleshoot :
1) Did you waited long enough to polymerize the gel ? - Partially polymerized gel can cause the wavy migration pattern.
2) Did you flush the wells before loading the samples? - Agarose or polyacrylamide/urea left in the well may interfere the migration and shape of the bands.
Run the gel at constant voltage, not more than 100V. higher voltage may cause streaking .
Parul
Thanks Gary! Yeah it might be the buffer because its not fresh and i did not checked the pH even.
Yes Parul i did waited for more than half hour to polymerize my gel.
But i did not flushed them, i will try next time.
Thanks for your suggestion.
Some times if you reuse buffer more then three times you could get same sort of results.
Ishwar, apart from preparing fresh buffer, I would suggest running the DNA samples at a lower percentage Agarose gel (0.4%) and less voltage (40 volts) for longer (like 4 hrs) ensuring that the DNA does not run off the gel.
Good luck!
As Parul, I think that the polymerization time was not enough. Maybe a very hot temperature of the room makes that you need more time. Maybe the agarose was not totally disolved... I would discard voltage, I use 120 or 150 and never had any problem.. For sure it is your gel, not your samples as the ladder looks bad as well. Good luck!
Clean up tank, use 1X TBE, change agarose, prepare fresh gel 1.2% in 1XTBE. Prerun the gel @60V for 10min, Load your sample and leave it for 2 min, then run it @60V. Hope you will find solution
It seems Purity is the problem. Check the uniform voltage supply.
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