need help in isolation and quantification of DNA

[carol]

I am writing a laboratory report on isolation and quantfication DNA from corn using gel electrophoresis. and I have to discuss each step in the procedure. Can you give me a link to a good reference for this? I can't seem to find one.

[Bluenoise]

Any protocol for isolation of DNA from plants should work. No need for it to be corn specific.

Look up CTAB plant genomic DNA extraction.

An example follows.

http://www.cilr.uq.edu.au/UserImages/File/Plant%20Genomic%20DNA%20Extraction%20by%20CTAB%20_2__Fiona.pdf

 

Same goes for electrophoresis. Just search for "DNA electrophoresis protocol" and you should be good to go.

 

 

Oppps sorry these aren't good references. I miss read your orriginal post. I may look one up when I have the time. Might want to try looking in the molecular cloning series of books.

[CharonY]

Are you seperating whole genome DNA?

Usually one need pulse field gel-electrophoresis for seperating DNA of that size.

Actually one usually quantifies DNA with a spectralphotometer. On gels one can use mass markers and densitometric measurement. I am not sure how good that works for chromosomes, though.

[Bluenoise]

Well you cant really hope to seperate individual chromosomes, but it works alright for a rough estimate. Also most genomic extraction methods don't typically result in full length chromosomes, they usually fragment into large chunks.

Though quantification isn's very acurate with gel electrophoresis as compared to spectrophotmeter, there are a couple advantages. You get some indication of the quality of your DNA or if there's a large amount of RNA contamination. Plus you don't typically waste as much sample quantifying it. ~1ug necessary for an acurate reading by spectrophotometry (assuming you only need to make one dilution) compared to ~20ng for electrophoresis.

[CharonY]

Well, I agree with the first part, but have to disagree with your assessment of photometers. Those that allow wavelength scans also allow an (rough) estimate of contaminations (in many protocols RNAse steps are involved anyway, protein contamination might be important). And regarding the amount, with special cuvettes you can use very low amounts of DNA. With the usage of newer machines (I like the NanoDrop for instance), you can go down to 1 µl of volume and few ng of amount for measurements.

[carol]

I din't want to be a bother, but can you give me the roles of reagents such as sodium acetate, chloroform, ethyl alcohol, isopropanol, Tris EDTA, ethidium bromide, the buffer solution added before grinding? I also want to know what can be done if there is no liquid N? Should the tubes be placed in ice? I just want to make sure.

[Bluenoise]

I don't have patients to tell you what each of these does. But I will tell you that the procedure will work pretty well even if you don't grind the material in liquid nitrogen. It might be harder to grind, and you might get some DNA degradation, but it should be alright for most purposes, as long as you don't take way to long with the proceedure.

[carol]

I'm done with the report. Thanks for all your help:)