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Principles behind DNA RNA protein isolation and purification


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#1 davince

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Posted 08 April 2009 - 01:59 PM

Hello everyone.

I know the questions i'm going to ask are probably stupid, but i really need to ask because i had used the words i knew to find anything that could lead to an answer.

What are the physical theories behind the isolation process and purification process of DNA, RNA and proteins?
What are the staining methods now we have used in staining DNA RNA and proteins specifically or non-specifically?

I tried to find a book that talks specific for these things but i couldn't find any.
(i hope it's only because i'm not using the right words googling.)

I'm an undergrad, and though i've taken classes in molecular biology and lab courses, i still have poor understanding on the why part.
everyone just does things by protocols with sometimes a little adjustments, and i was no different, just follow the protocols.

but i really need to know why.

What are the theories behind the purification and isolation of DNA, RNA and proteins from like say cell lysate?
i know there are alot of things, ethonal precipitation of nucleic acids, silica binding, phenol-chloroform purification, alkaline lysis for plasmids, protein precipitation by salting out.
Some of them have entry in wikipedia, but some i think not
but i really want to understand the theory behind it
why the techniques can isolate one and not the others
i know many uses the theories in physical chemistry, but why the particular chemicals?
(or is it because i didn't learn biochemistry well?)


Another problem didn't concern me before i'm writing on a one of the paper required in a lab course. We were silver staining polyacrylamide gels, and the target is RNA.
After reading some more papers, i found out that silver stains actually stains all of them. DNA, RNA, lipids, proteins, a bunch of things.

Are there any books talking about the staining methods and principles of staining DNA, RNA and proteins specifically or non-specifically?

what i know is that EtBr, SYBR Green can stain DNA, dsRNA
Commassie blue can stain proteins
and silver stain actually stains for all of them.
i usually though silver stain only stains proteins... but later found out that this was actually not true.



I really hope you can help me out
it's killing me :P

thanks alot
I'm really appreciated

#2 Ahrenhase

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Posted 08 April 2009 - 06:22 PM

All these isolation techniues simply take advantage of the atomic makeup of nucleic acids and proteins.
Type in "How ethanol precipitation DNA works" it'll come up. It all has to do with optimal pH's, temps, ion content, molecular bonding, solubility, and things like that. Some of the mechanisms aren't even known completely for sure.

Have you taken all you chem courses(organic, biochem)? These help.

#3 davince

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Posted 09 April 2009 - 03:15 AM

All these isolation techniues simply take advantage of the atomic makeup of nucleic acids and proteins.
Type in "How ethanol precipitation DNA works" it'll come up. It all has to do with optimal pH's, temps, ion content, molecular bonding, solubility, and things like that. Some of the mechanisms aren't even known completely for sure.

Have you taken all you chem courses(organic, biochem)? These help.


yes i've taken a few chem courses like general chemistry organic chemistry and biochemistry.
but the courses i took were in my opinion poorly designed
for example, organic chemistry course i took is a one-semester course, really little was learned then.

(though i don't know if that is the way it is in every college around the world....i hope not)


thanks alot...i'll look into the how..works keywords
:rolleyes:




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