Gel Electrophoresis SAC - Help Please :)

[sillysmile]

Perhaps it's there because the retriction enzymes didn't cut all of the dna, and missed just a small few which caused them to be larger, and thus not very far travelled. The light shade could be explained by the small amount of the dna that restriction enzymes had missed. This is a reasonable possiblity I believe.

[stonecold]

thanks for your help, but the problem with that is the smallest fragment in the sample is meant to be over 5000bp, which is quite large and is the bottom one in the lane.  this 'unexpected' one looks significantly smaller, and can't belong to that fragment.

is it possible that a mutation occurred in the upper fragment, causing a new recognition sequence which the enzyme cut during electrophoresis, resulting in the smaller fragment seen?

[sillysmile]

it's possible, but to be honest, the chance of a spontaneous mutation is incredibly small. Did you get any background info about the DNA, that you might have overlooked? are you sure the DNA wasn't contaminated by anything?

[TrueLight]

which fragment are you talking about? the first lane? second band?... no idea maybe there wasn't enough dna in the sample and so it would have separated but because there's less dna its just less dense

[stonecold]

which fragment are you talking about? the first lane? second band?... no idea maybe there wasn't enough dna in the sample and so it would have separated but because there's less dna its just less dense

yeah, first lane, second strand, which is way thinner than the rest, and according to the fragment map thing we got, it shouldn't be there...

[TrueLight]

why shouldn't it be there is there meant to be a fragment between those two brighter bands? there isnt even a standard to compare... maybe its the reason i stated above or maybe there a restriction site close to the two sites that make the large fragment. and so it might have cut at the different areas or something something........lol

[stonecold]

there is a standard sorry lol.  i have it with me. 

Lane 1 is meant to have bands at:
16841 bp
7233 bp
6770 bp (meshed with next one)
6527 bp
5672 bp (meshed with next one)
5505 bp

The random, thinner strand is in between the first two as you can see.

If incomplete digestion occurred, wouldn't the band be fatter?

[TrueLight]

if you get incomplete digestion it means you have less fragments but doesn't mean the bands are wider, if the fragment is long you might get brighter bands.but yeah maybe it didn't separate properly...


ps. if this doesn't make sense its okay i might just be hallucinating since i had no sleep last night doing a stupid prac report lol

[stonecold]

okay, thanks everyone. i'll try and write something down that doesn't sound ridiculous.

sorry for all the questions.

[TrueLight]

lol yeah but the spontaneous mutation to make a restriction site is very very unlikely. so i probably wouldnt put that explanation in

[stonecold]

sweet.  i got rid of it.  thanks again!!

[masonnnn]

not sure if this is too late but the dna could have been contaminated before pcr resulting in the unexpected band being produced...
but even that you'd think would produce multiple unexpected bands, not just the one.
doesn't seem to be much to explain that...

[PeterVest]

Gel electrophoresis is known as way to separate and analyze macromolecules (DNA, RNA and proteins) based on their size and charge. It is generally used in clinical chemistry, biochemistry and molecular biology. It uses a gel which acts as an anticonvective medium while electrophoresis i.e. the charged particle movement in an electrical field. This process can be used in the separation of nanoparticles too.

[pi]

Um, ok.

This thread was from 2010, please don't post on old threads unless it's completely necessary.