Biotech Lab Protocols, Tips & Tricks

PURPOSE: To digest given DNA sample using restriction enzyme EcoR1.

REQUIREMENTS: DNA ,Distilled water, Restriction enzyme ,Enzyme buffer, Heating block, Electrophoresis assembly

PRINCIPLE: A Restriction digest is a procedure to prepare DNA for analysis or other processing. The enzymatic technique can be used for cleaving DNA molecules at specific sites ,

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ensuring that all DNA fragments that contain a particular sequence have the same size,furthermore,each fragment that contains the desired sequence has the sequence located at exactly the same position with in fragment .Restriction enzymes are the enzymes that cut double stranded DNA by making two breaks, one through each of the phosphate bones of the double helix. The enzyme does this with out damaging the bases of the DNA .Although the enzymes breaks the DNA,the chemical bonds can be reformed by other enzymes known as DNA ligases. There fore,restriction fragments of DNA from different chromosomes or genes can be ligated together,providing their ends are complimentary

PROCEDURE:

1.Restriction mixture was calculated according to the standard calculation.

2.Group wise different restriction enzymes were assigned&accordingly master mixes were prepared

3.One hour incubation was given to these reaction mixture at 37degree C

4.Gel electrophoresis was performed next in order to analyze the restriction digestion sample

5. In each well,13ul of the restriction digested sample & 2ul of loading buffer(total of 15ul of solution was added to the gel)

6. Along with the sample, there were two wells in which DNA ladder was added &one of the well had uncut DNA

REACTION MIX:

RESULT: Restriction digestion of given DNA sample was carried out using EcoR1

INFERENCE: Under UV trans illuminator, two distinct band was visualized. one was nicked DNA plasmid (heavier one)& other of insert (lighter one)were observed on the gel.