Gel Electrophoresis SAC - Help Please :)

[stonecold]

Can someone please tell me how:

-Stains like methylene blue are able to show the DNA as bands in the gel and;

Also, when you are counting the number of bands in a electrophoresis gel, you don't count the band at the well right?

Thanks.

[sillysmile]

I believe that the stains bind to dna, and no you don't count the band in the well.

[stonecold]

Thanks.  Yeah, I just don't know how it binds.  Do I have to go into detail?

[sillysmile]

yes, that would probably be a good idea, tbh I don't know how methylene blue bins to dna. I know of another stain, called ethidium bromide which binds inbetween base pairs.

[stonecold]

Anyone know how the stain works?

Also, what are some of the reasons for certain bands not being visible, or for unexpected bands in the lane?  One of the questions asks this, and there is a band which is distinctively smaller and lighter than the others in the same lane, yet it is in between two bands which are quite large and show very strongly.

I can upload it if it makes things easier.

[stonecold]

This is what we were given. First lane is supposed to have 6 fragments, however has an 'unexpected' fragment.  Second is supposed to have 6 fragments, and the third is supposed to have 8.  The final lane is of a the piece of DNA which hasn't been cut, so it is one big piece.

Oh, and the two outer lanes are empty btw...

[sillysmile]

ok, did you guys use a dna ladder (a.k.a standard) for comparison? and I believe the reason why lanes 2 and 3 both appear to have less bands then dna fragments, is because there are probably some dna fragments that are of similar base-pair length (or size) for e.g. pretend there are 4 fragments in a particular lane, 1st is 4500bp (bp=base-pair length),  2nd is 4600bp 3rd is 2000bp 4 is 1000bp. Since the first two fragments are so similar in base-pair length, they will travel at a very similar speed, and consquently be very close together, resulting in an inability to differentiate between them as the bands have blended into one. In regards to the first lane in your sac, perhaps their is an additional band because, the dna fragment was different in its base sequence then what it was supposed to be, which means that the restriction enzyme would have cut at different points, and perhaps ending up with a greater number of fragments, or alternatively a larger size or a lesser amount of fragments. One other possible reason could be that the DNA was contaminated with other DNA. 

[Stroodle]

Can someone please tell me how:

-Stains like methylene blue are able to show the DNA as bands in the gel and;

Also, when you are counting the number of bands in a electrophoresis gel, you don't count the band at the well right?

Thanks.

Methyl blue has a positive charge, enabling it to bind to the negatively charged dna fragments, and I don't think you count the bands in the wells.

[sillysmile]

btw the darker the band is, the more dna is in that region.

[jasoN-]

If you're bored, there's an interactive experiment with gel electrophoresis being applied in this website (it's educational)
http://learn.genetics.utah.edu/content/labs/gel/index.html

[TrueLight]

why is it supposed to have 6 or 8? what did are the samples you put in the gel?
yeah your supposed to have a standard ladder too

maybe you didn't run the gel long enough to seperate them out or yeah maybe they are have the same size

[stonecold]

thanks guys.  this was a worksheet at the end of our sac.  i didn't run the electrophoresis.

and there is no ladder, just a table outlining the number of pieces which each restriction enzyme cut the DNA into...

[TrueLight]

oh lol

also maybe there wasn't complete digestion of the dna

[stonecold]

thanks all.  does anyone know how the unexpected band came about in the first lane?  i'm assuming it is the lighter, second one from the top.  we have to specify it and explain why it may be there.

[stonecold]

i'm trying to explain why it is there, but it's hard.  it appears thinner, which means you would expect it to be further down. something weird must have happened, because even if the sample was contaminated, you would expect it to have moved the furthest, being the smallest fragment. also all the other bands which are meant to be there are present, a couple are just bunched up together and not visible.

is it possible that that fragment was only digested in the final moments or something?  even then that doesn't explain a great deal, because as i've said, i've already accounted for all the fragments that should be there.  that one is far to small.

maybe a mutation occurred during electrophoresis, creating a new recognition sequence, and it was cut, but didn't migrate to where it should have because it happened in the later part of the experiment.  I dunno, i'm just making stuff up.  this sounds kind of plausible haha...