Need help, what's wrong with my DNA gel? - (Feb/06/2008 )
Hi, all,
I ran a 4-20% TBE gel(premade gels from invitrogen) with my sonication samples with homemade TBE buffer and sample loading buffer from qiagen. I ran gel at 200voltage. As you can see from the pic, the DNA seemed to shift to one side. I'm wondering what's wrong with my system. Have I used a voltage that's too high?
Thanks a lot!
did the running start from the bottom of the gel as shown on this pic?
I am not quiet certain what when wrong, but it look like uneven loading. Tilted gel box? Hmm.....
However I would say 200V is too high. I don't know how big your gel is (bigger gels can take higher voltage), but my guess is that your gel is on the small side. And what fragment size are you trying to visualise?
The sample fragment size seem to large for 100bp ladder. Are you looking at genomic DNA? In which case it is best to check for DNA degredation by running a 1% gel. Sonication should leave you fragments in the 5 -10kb range.If the fragments are in the 2kb range and below your DNA is degraded.
Try 80V. (I tend to like running things slowly) And take care not to stab/poke your gel when adding the sample. And make sure that the table is actually horrizontal and not tilted. Phyically tilting the gel box could actually account for the effect.
And make sure your electrode are actually parallel to one another. If they aren't well, the electric field won't be straight and the DNA won't run straight.
However I would say 200V is too high. I don't know how big your gel is (bigger gels can take higher voltage), but my guess is that your gel is on the small side. And what fragment size are you trying to visualise?
The sample fragment size seem to large for 100bp ladder. Are you looking at genomic DNA? In which case it is best to check for DNA degredation by running a 1% gel. Sonication should leave you fragments in the 5 -10kb range.If the fragments are in the 2kb range and below your DNA is degraded.
Try 80V. (I tend to like running things slowly) And take care not to stab/poke your gel when adding the sample. And make sure that the table is actually horrizontal and not tilted. Phyically tilting the gel box could actually account for the effect.
And make sure your electrode are actually parallel to one another. If they aren't well, the electric field won't be straight and the DNA won't run straight.
Thank you. You are right, I have a minigel acually. I'm seeing a fragments between 200 to 4000bp. These are chip samples so they are supposed to be best if below 1kb.
I think I'll try your suggestions, run the gel slowly. I believe I did not stab my gel but I need to be more careful. i did not really look carefully whether the table or the gel boxes are tilted, that's a great suggestion!
No, I run gel from the top of the gel, sorry that I did not make it clear in my post. You can also tell from the seperation of the marker that the gel was run from top.
I know what's wrong. There is no DNA on your gel.
Really, I'm serious. There may be a a little at the top but I don't really see any DNA.
It's really hard to visualize something that simply isn't there.
My advice to to revamp your DNA purification protocol so you actually isolate DNA that you can see on a gel and then work on the sonication. You're probably losing it during the phenol/chloroform step (?).
Mikew is right. There is a smear. Not a proper band. Run your gel at lower voltage, and make sure the electrodes are properly connected.
should it not look like a smear? The DNA has been sonicated. And the range of fragments being looked at by COCOMILK is below 1kb.
i apologize. I didn't realize it was sonicated sample.
Yes, the sonicated DNA would run as a smear. However, the input
does not give a visible band. The input should be a big bright blob. It is not. Therefore the DNA isolation is very inefficient.
If you take a small amount of DNA that is sonicated it will smear-out and will not be visible because the quantity
is below the detectable range. So, yes, there is DNA on the gel, but not enought to see, so for all intensive purposes there is no DNA on the gel.
Even 1 microgram of genomic DNA will appear as a large blob on a gel.
Maybe more DNA should be loaded.
Try loaded the whole DNA sample from ~ 2 million cells.