Thursday, November 1, 2012

Extraction of Plasmid DNA - Principle behind usage of various reagents

Hi,
Have a good time :) Last time I had explained you the procedure for "electroporation" and now with those electroporated cells, you can extract the plasmid DNA.

I'm not going to share the exact protocol here, but the mechanism behind the protocol we use in our lab.

Generally, the following reagents are used for the extraction of plasmid DNA.,
1) STET buffer
2)Lysozyme
PUC19
3)RNAase
4)Phenol chloroform
5) Alcohol
6) Sodium acetate

If you need the protocol, kindly mail me I'll send you the  protocol. Now, let us discuss the principle behind the usage of each of these reagents.

STET buffer - contains Sucrose which helps in maintaining osmotic pressure; Triton X which helps in cleaving the cell wall; EDTA which acts as a chelating agent; Tris HCl is used as buffer.

Lysozyme - cleaves the cell membrane and wall;

RNAase- As  the name indicates, it cleaves RNA

Phenol chloroform (buffer saturated- pH 8) - Phenol denatures proteins, Chloroform prevents oxidised phenol from binding with DNA, it also takes care of lipids and carbohydrates.

Alcohol and sodium acetate - Helps in washing and precipitation of DNA; Sodium acetate provides high salt content, which is necessary for the precipitation of DNA.

 Hope this helped you, any doubts? Then, mail me, happy to clear your doubts (if I can, as I'm not an expert)

Wish you a very happy day :)

Bye...!


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