Wednesday, 25 November 2009

GGS LIVE - DNA digest

Method: DNA digest

About:  Simply saying DNA digestion is cutting the DNA into pieces (before you go further have a quick look at this animation about restriction endonucleases McGraw Hill Animations - Restriction Endonucleases). What are the purposes of DNA digestion? They can be different like analytical  (to see if the DNA is what we thing it is), preparative (to extract specific DNA sequence) or others (for example southern blot, cell transfection or DNA based assays).

What: Digestion of the plasmid DNA with either single, double or no cutting enzyme.

How: By incubation of the digestion reaction containing DNA with appropriate restriction enzyme.

In our experiment we are going digest our DNA sample with two different enzymes. One of them (BamHI) cuts only once in our sequence but the other restriction enzyme (HindIII) digest our DNA at two different sites. As a control for the experiment we will incubate DNA sample reaction containing no enzyme. Our DNA is a plasmid DNA of around 5,5kb in size (see picture below):

Regular digestion reaction is composed of:
                              x microl of DNA
                              5microl of 10x Buffer
                              5microl of 10xBSA
                              0,5 - 1,0microl of enzyme
                              Up to 50microl of high pure water
DNA - our sample (amount of the DNA used depends on assay)
Buffer - delivers appropriate conditions for the enzyme
BSA  - presence of other proteins (Bovine Serum Albumin) increase enzyme stability
Enzyme - no comments:)
Water - no need to comment:)

Usually we incubate the reaction at 37 degrees C for ... (time of incubation depends on amount of DNA or enzyme used). After the digestion an agarose gel electrophoresis is performed to see if the digestion occured (see below).

As you see at the picture above as expected, single cut and double cut of our DNA results in single 5,5kb band or two 2,5kb and 3,0kb bands, respectively. Remember that plasmid DNA can exist in different conformations, relaxed or supercoiled. Supercoiled form of DNA (in which plasmid DNA usually exists) is more compacted and that is way uncut DNA appears as a smaller band on the agarose gel (it migrates faster than expected from its size).

This is it:)

I hope you enjoy it:)


GGS Team

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