Digesting the DNA
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:
(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.
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.
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'
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