Isolation of Bacteriophage DNA and study of its Hind III digestion pattern

[ps2id id=’background’ target=”/]

BACKGROUND

Bacteriophages are mainly composed of high-quality proteins encapsulating a DNA or an RNA genome, and possess a very simple or intricate structure. Their genomes may show as little as four genes or as many as hundred genes. Inside the bacterium replication of the phage occurs following the injection of their genome into the cytoplasm. So, the basic objective of this experiment is isolation of bacteriophage DNA and studying its HIND III digestion pattern.¹

[ps2id id=’requirements’ target=”/]

REQUIREMENTS

Reagents:       Phage lysate

Nuclease solution

Precipitant solution (Take a clean bottle and add 110 ml dd H₂O and 35 g NaCl, and mix it properly so that it completely dissolves. To it add 60 g of PEG- 8000, then put the bottle cap and shake it. Incubate the bottle in a 50-60^oC water bath for a duration of 2 hours, shake it occasionally. Remove it and let it cool down to room temperature along with occasionally shaking. This solution should be clear and little turbid. Add ddH2O to 200ml and store it in room temperature.)

Resuspension buffer (5 nM MgSO₄ dissolved in water)

80% isopropanol

1X Buffer

10 mM Tris-HCl

50 mM NaCl

10 mM MgCl₂

1 mM Dithiothreito

Sterilized water.

Materials:      Centrifuge tubes of 50ml

Sterilized microcentrifuge tube of 1.5ml and 2ml

Syringes 3cc

Heat block with temperature 80^oC

50 ml centrifuge tubes

Sterile 1.5 and 2 ml microcentrifuge tubes

3 cc syringes

Heat block, 80 ºC

[ps2id id=’procedure’ target=”/]

PROCEDURE

At first prepare the phage lysate either by plate lysate or by liquid culture methods. By filter sterilization clarify the lysates. You can keep the lysates either in broth or in lambda- dil. Now take a clean 50 ml centrifuge tube and place the lysate in it and add 0.5 microlitre of nuclease solution per ml of lysate. Then keep the lysate in incubator at a temperature of 37^oC for about 30 min. Now add the precipitant solution to the lysate in a ratio 1:2 precipitant:lysate. Mix it properly by inversion. Incubate it on ice for about 60 min. When it is kept in temperature of 4^oC, best precipitation occurs. Most of the phages remains stable in this condition for several days. Now put the precipitated phage lysate in centrifuge and spin it at 10000 × g, at a temperature of 4^oC for about 10 min. Now carefully put out the supernatant and retain the pellet.^2-4 The pellet may be either opaque or transparent or may also get spread to the wall of the tube. Now suspend the pellet in 500 microlitre of resuspension buffer and with the help of a pipette mix it. Now transfer this resuspended phage into a 1.5ml of microcentrifuge tube and centrifuge it for about 5-10 sec to pellet down any insoluble particles. Now transfer the supernatant to another microcentrifuge tube. Now properly resuspend the purification resin and add about 1 ml of this resin to the phase suspension. Mix it properly by inverting the tube for about 5-6 times. For each DNA label a 1.5 ml microcentrifuge tube and keep it in a tube rack. To it add minicolumn into each tube. From a 3ml syringe remove the plunger, attach the syringe barrel to the minicolumns in the rack and left them standing in the tube rack. Place the plunger on a clean paper towel. Now with the help of pipette transfer the resin or the lysate mix into the syringe. Now hold on the syringe above the waste beaker, insert the syringe barrel and put the resin into the minicolumn. Keep pressing till all liquid has been put out through the resin. Now detach the minicolumn from the syringe and keep it back into the microcentrifuge tube. Now put out the plunger from the syringe and attach the syringe barrel to the mini column. Now clean the minicolumn by addition of 2ml of 80% isopropanol to the syringe. Again, hold the syringe above the waste beaker, insert the syringe barrel and push the isopropanol through the minicolumn. Keep it pressing until and unless all the liquid has been put out through the resin. Now remove the syringe from the minicolumn and throw the syringe. Now take a new 1.5 ml microcentrifuge tube and cut its lid off, label it and place the minicolumn into it. Keep it in centrifuge for about 2 min and rotate at 13000* g, to dry out the resin. Now keep the tube and the minicolumn into the microcentrifuge. Pipet 100 microlitre, add another 100 microlitre of hot water and mix it by spinning. Now take a new 1.5 ml microcentrifuge tube and label it properly. Now put the eluted DNA into the new tube. Throw the minicolumn and close the lid and store the DNA at – 20^oC.

The HIND III restriction enzyme consist of the following buffer components 1X Buffer 10mM Tris‐HCl   50mM NaCl 10mM MgCl2 1mM Dithiothreitol pH 7.9. Usually you can use I unit to digest 1 microgram of DNA in an hour. Before doing digestion, you should preheat the heating block to about 37oC. In an ice bucket you will keep five tubes sample tube containing the DNA, a tube with 10X buffer, a tube with some water and a tube with some enzyme at a concentration of 10units per microlitre. Now set up your digestion in a 1.5 ml Eppendorf tube. Mix water, 10X buffer and DNA and mix it properly by pipetting it up and down. Then mix the enzyme properly. In a small centrifuge spin it for a very brief time so that the sample is collected in the bottom. Now place it in the preheat block. This reaction will take minimum 1 hour. Then add 5 microlitre of 7.5M ammonium acetate and 100 microlitre of 100% ethanol to each tube and mix it properly by inverting it for many times. Centrifuge it and suspend the DNA in 25 microlitre of TE buffer, and load 5 microlitre of each sample in agarose gel.

[ps2id id=’conclusion’ target=”/]

CONCLUSION

It is one of the best proven method for isolating the bacteriophage DNA and study its digestion pattern.

[ps2id id=’references’ target=”/][ps2id id=’1′ target=”/]

REFERENCES

1. Alwine JC, Kemp DJ, Stark GR. Method for detection of specific RNAs in agarose gels by transfer to diazo benzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci USA. 1977; 74:5350–5354.
   
2. Bautz FA, Bautz EKF. Mapping of deletions in a non-essential region of the phage T4 genome. J Mol Biol. 1977; 128:345–355.
   
3. Carlson K, Nicolaisen B. Cleavage map of bacteriophage T4 cytosine-containing DNA by sequence-specific endonucleases SalI and KpnI. J Virol. 1979; 31:112–123.
   
4. Depew R, Snopek TJ, Cozzarelli NR. Characterization of a new class of deletions of the D region of the bacteriophage T4 genome. Virology. 1975; 64:144–152.