Digesting the DNA

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What do we mean by 'Digestion'?

Digestion of DNA is a process carried out just to be able to look at DNA (like an analytical gel) or, like in this case - cut a band out of the gel for further treatment (a preparative gel).

Typically, a band is easily visible under UV light in an ethidium-bromide-stained gel if it contains about 20nanogram of DNA. 

Digestion can be done in 2 ways:

1.Double 2.Single

Ethidium Bromide:

(EtBr) is an agent that is used as a fluorescent tag to see the DNA.When exposed to ultraviolet light, it will fluoresce with an orange color, intensifying almost 20-fold after binding to DNA.

Restriction nuclease:

An enzyme which cuts DNA.

Restriction endonucleases such as EcoRI recognize specific palindromic sequences and cleave a phosphodiester bond on each strand at that sequence. After digestion with a restriction endonuclease the resulting DNA fragments can be separated by agarose gel electrophoresis and their size can be estimated.

Palindrome/Palindromic Sequences:

A palindrome is a word that is the same forward as it is backward like the word MALAYALAM. Certain sequences of DNA are the same whether they are read 5' (five-prime) to 3'(three prime) or 3' to 5'. These sequences are called palindromic.

5'- G  A  A  T  T  C -3'
3'- C  T  T  A  A  G -5'

Double Digestion:

When you are digesting your DNA with two (or more) enzymes. In such cases, you have to make sure to use the buffer that will be most compatible with all the enzymes. Enzyme buffers are specifically formulated to provide the salt concentration for optimal enzyme activity. There should not be too much salt in the digestion because too salt salt will inhibit enzyme activity.

  • (Lysis= K112808)

pBAD- promoter Lysis-big construct- gene for cell lysis Gel image of released pBAD(I0500), after digestion with EcoRI+PstI:

Each pBAd and Lysis has 4 restriction enzymes E, X,S, P.

pBAD is digested with EcoRI +SPeI Lysis is digested with EcoRI + XbaI


        Plasmid   Concentration   260/280
        IO500(P)   1790.5µg/µL     1.92
        Lysis(L)   2324.4µg/µL     1.91

For Short Digestion

                DNA = 2µL
 ECORI + Pst1 E1+E2 = 0.5µL + 0.5µL
                BSA = 2µL
             Buffer = 2µL
                 MQ = 13µL
              Total = 20µL

Time taken = 1.5 hours