Strange question regarding DNA in gel-slice... - (Nov/11/2005 )
QUOTE (beccaf22 @ Nov 17 2005, 03:38 PM)
Hi, so I think you are still seeing the same thing... Yes the EtBr gets into the gel, what happens is the DNA nearest the outside of the gel is taking the EtBr up...
Lets take the "bottom of the well" band... The EtBr migrates into the gel far enough to reach this DNA (obviously
) What happens there is that the EtBr is getting intercalated into these DNA strands, so the DNA there is "soaking up" the EtBr and doesn't let it pass by to intercalate into the DNA in the middle of the gel. So it isn't that EtBr cant get into the gel, just that the DNA nearest the outside of the gel is like (if you will give me some metaphoric leeway) a sponge, you have to saturate this DNA with EtBr before it can get intercalated into the DNA at the middle...
I think this is probably what is going on, I hope this makes sense to you... You may ask, as I am wondering now, why the DNA in the middle of the band isn't picking up EtBr from the front and back side (ie the top and bottom of the lane), but this I just don't know...
Maybe someone else has an idea...
Lets take the "bottom of the well" band... The EtBr migrates into the gel far enough to reach this DNA (obviously
) What happens there is that the EtBr is getting intercalated into these DNA strands, so the DNA there is "soaking up" the EtBr and doesn't let it pass by to intercalate into the DNA in the middle of the gel. So it isn't that EtBr cant get into the gel, just that the DNA nearest the outside of the gel is like (if you will give me some metaphoric leeway) a sponge, you have to saturate this DNA with EtBr before it can get intercalated into the DNA at the middle... I think this is probably what is going on, I hope this makes sense to you... You may ask, as I am wondering now, why the DNA in the middle of the band isn't picking up EtBr from the front and back side (ie the top and bottom of the lane), but this I just don't know...
Maybe someone else has an idea...
Hi,
Well, it seems like your theory about the EtBr getting "stuck" in the DNA far up and far down in the lane is correct. I ran a gel some days ago, and stained it as usual in EtBr. I cut the bands and tilted the gel slices 90 degrees and could, as usual, see distinct bands at the bottom of the well and far up. I had two samples with rather different volumes (same amount of DNA) and while the bottom band was similar in the two samples the localisation of the uppermost bands were different with the "small-volum-sample" having its band somewhat lower than the "high-volume-sample" (not very suprising, since I just added the samples to the wells without making any attempts to spread them into the whole well volume, I just let them "fall into the well" and that's it). Then I took the gel slices and placed them in the EtBr. It suddenly became very clear that my removal of "empty gel" between the upper and lower band is not very intelligent... The area between the bands was clearly stained, just as intense as the original bands. So yes, the DNA is there, it's just not so easy to see...
-sonjaa-
QUOTE (sonjaa @ Dec 18 2005, 12:48 AM)
So yes, the DNA is there, it's just not so easy to see...
thx for performing that experiment
-Kersten-
That was a very smart move:) Is there something we still need to answer after that..?
-cheeztoast-