Another gel electrophoresis question

[Gamewizard]

Another question i need a bit of help on.

 

The Total length of Calf Thymus DNA is several million base pairs long, how is this reflected in your experimental observations?

 

ok so i have a photograph of the gel i made. and i can see straight long lines of DNA.

[dttom]

do you have markers, comparing your band with the marker should tell the length of DNA.

[Gamewizard]

No i dont think i have markers

[ecoli]

Otherwise known as a DNA ladder, which fragments of known sizes so you can compare.

 

I don't know of any gel that can discriminate a DNA fragment that's a million base pairs, however. Did you further fragment this gene?

[Gamewizard]

No we didnt further fragment it, and they havent given us any fragments to compare sizes with.

[ecoli]

No we didnt further fragment it, and they havent given us any fragments to compare sizes with.

Maybe you can explain exactly what you did do, then?

[Gamewizard]

Ok, we just did the simple electrophoresis procedure.

 

Made agarose gel, then connected it to the power supply, added buffer in it, loaded 4microlitre of the DNA samples in it and then ran the gel for about 30 minutes.

then they were stained with ethiduim bromide and viewed under ultraviolet light. And then we got the photograph showing the DNA bands.

[CharonY]

It is a trick question (of sorts). If the DNA was whole, you would essentially see a more or less defined band at the very top of the gel. It would not move much due to the size. Also, no marker would help you at that size (just keep in mind that we are talking about whole chromosomal DNA here).

But I suppose you may be seeing more a less a smear from top to bottom rather than defined bands. What do you think this could be?

[Gamewizard]

It is a trick question (of sorts). If the DNA was whole, you would essentially see a more or less defined band at the very top of the gel. It would not move much due to the size. Also, no marker would help you at that size (just keep in mind that we are talking about whole chromosomal DNA here).

But I suppose you may be seeing more a less a smear from top to bottom rather than defined bands. What do you think this could be?

 

Yes you are right, from the photograph i can see smears from top to bottom they are not defined bands as compared to others.

 

Thats what i cant figure out. is it because its the whole chromosomal DNA? and possibly the whole double helix structure of DNA?

[CharonY]

Nah, it may be a bit hard to figure out. So here is the answer:

Long DNA molecules are mechanically unstable. If insufficient precautions are taken normal liquid handling alone is usually sufficient to shear it.

So what you see is that the chromosomal DNA has been sheared (plus, depending on the extraction protocol other forms of degradation may have occured). So what you see is a broad distribution of lots and lots of different chromosomal DNA fragments with the intact one being somewhere on top.

[Mr Skeptic]

nevermind

Edited March 25, 2010 by Mr Skeptic
what CharonY said.

[Gamewizard]

Thank you so much CharonY.