VPB 321: Animal Biotechnology (2+1)

RESTRICTION ENZYME ANALYSIS

Aim

• To study the restriction enzyme pattern of  DNA.

Material required

• DNA, restriction enzymes (EcoRI, BamHI, HindIII), restriction buffer, distilled water, loading dye, Tris/Borate/EDTA buffer, agarose, ethidium bromide, 0.5 – 10 m l micropipette + tips, 1.5 ml tubes, aluminium foil, beaker, test tube rack and water bath.

Procedure

• Use permanent marker to label four 1.5 –ml tubes, in which restriction reactions will be performed:
  • B = BamHI; E = EcoRI ; H = HindIII ; - = no enzyme.
• Use matrix below as checklist while adding reagents to each reaction. Read down each column, adding the same reagent to all appropriate tubes. Use a fresh tip for each reagent. Refer to detailed directions that follow.
• Collect reagents, and place in test tube rack on lab bench.
• Add 4 m l of DNA to each reaction tube.
• Use fresh tip to add 5 m l of restriction buffer.
• Add 1 m l of EcoRI, BamHI, and Hind III to appropriate tubes.
• Add 1 m l of deionized water to the tube labeled “ -”
• Close tube tops. Pool and mix reagents by pulsing in a microfuge or by sharply tapping the tube bottom on lab bench.
• Place reaction tubes in 37 ° C water bath, and incubate for a minimum of 20 minutes. Reactions can be incubated for a longer period of time.
• Run the samples on 0.8 % agarose gel and stain with ethidium bromide to visualize the restriction pattern of different restriction enzymes.
• Figure below gives the details about the action of BamHI restriction enzyme on double strand DNA

Questions

• Why is water added to tube labeled “-” in the procedure?
• What is the function of restriction buffer?
• How does ethidium bromide stain DNA?