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  Section: Plant Lab Protocols
 
 
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Methodology for Nucleic Acids

 
     
 
Cultivation of lambda (l) phage
 
Unlike bacterial cells, viruses have to be multiplied in living hosts. Bacteriophages are prepared by allowing them to infect and multiply on bacterial cells. Phage l is used as a vector for cDNA and genomic libraries construction.
 
 



Principle

Bacteriophage l is cultured in E.coli cell suspension. The phage is allowed to harbor and multiply till the complete lysis of bacterial cells. Then the bacterial cells are killed with chloroform and the phage particles are separated by centrifugation.
 
 



Materials

E. coli LE 392 or other suitable strains
Stock A phage (EMBL-3)
Chloroform
Pancreatic DNase and RNase
Sodium Chloride
Polyethylene Glycol (PEG 6000)
Cesium Chloride
Luria Broth
     Bacto-tryptone                                10g
     Yeast extract                                    5g
     Sodium chloride                             5g
     D-glucose                                        1g
     Water                                                1L
     Sterilize and use
SM Buffer
     Sodium chloride                             5g
     Magnesium sulphate                    1.2g
     1M Tris (pH 8.0)                              50mL
     Gelatin                                              0.1g
     Water                                                1L
     Sterilize and use.
 
 
Procedure
1.
Inoculate 1mL of overnight grown culture of LE 392 E.coli strain to 100mL LB containing 10mM MgCl2 and 0.2% maltose in triplicate. Grow for 2-3h till the OD 600 reaches 0.4.
2.
With the assumption that 10D600 = 8 X 108 cells/mL calculate the cell concentration.
3.
Centrifuge the cell suspension in autoclaved tubes at 4000 x g for 10 min at room temperature.
4.
Discard the supernatant and resuspend the bacterial cells in 2mL of SM.
5.
Add bacteriophage from the stock to a concentration of 5 X 108 phage/mL and mix rapidly.
6.
Incubate at 37°C for 20 min. Shake intermittently.
7.
To 125mL of LB containing 10mM MgSO4 and 0.2% maltose, add one mL of infected cells (Step 4) in 500mL flask. Incubate at 37°C with very vigorous shaking till it lysed. Disappearance of silkiness is the indication of complete lysis. It takes 7-9h for complete lysis.
8.
To check complete lysis, take 1mL of the culture from the flask and add few drops of chloroform. If it becomes clear proceed with the next step. If not, continue the incubation till complete lysis is achieved.
9.
Add 2.5mL chloroform to each flask and continue vigorous shaking for another 30 min at room temperature.
10.
Collect the supernatant and pool for virus particles.
11.
Bring the culture to room temperature and add pancreatic DNase and RNase, both to a final concentration of 1mg/mL. Incubate for 30 min at room temperature.
12.
Now, add NaCl to a final concentration of 1M (29.2g/500mL of culture). Dissolve by swirling. Let it stand for 1h on ice.
13.
Remove white silky mass of bacterial cells by centrifugation at 11,000 X g for 10 min at 4°C. Pool the supernatant and repeat this step.
14.
To the supernatant in a flask, add solid polyethylene glycol (PEG 6000) in small quantities with slow shaking to a final concentration of 10% w/v (i.e. 50g/500mL).
15.
Cool in ice water and keep it at 4°C overnight.
16.
Centrifuge at 11,000 X g for 10 min at 4°C and recover the precipitated phage particles.
17.
Resuspend the bacteriophage pellet by gentle shaking in 7mL SM buffer (i.e., 7mL/500mL of original supernatant).
18.
To this suspension add equal volume (7mL) of chloroform and mix thoroughly. Centrifuge at 1600 x g for 10 min at 4°C. Save the aqueous phase containing bacteriophage.
19.
Measure the volume of this phage suspension and add 0.5g/mL of solid cesium chloride. Mix gently. After completely dissolving cesium chloride, carefully layer this suspension onto cesium chloride step gradients that are prepared in cellulose nitrate centrifuge tubes (refer note below for the preparation of CsCl step gradient).
20.
Centrifuge using SW 27 rotor at 25,000rpm for 3h at 4°C. A bluish band of bacteriophage particles will be visible at the interface between the 1.45 and 1.50g/mL layers.
21.
Collect the band of bacteriophage particles using siliconized Pasteur pipette. Store at 4°C in tightly capped tube.
 
 
Notes
 
Step Gradient
Prepare cesium chloride solutions of 5M and 3M separately in distilled water. Transfer 10mL of 5M solution to the bottom of cellulose nitrate centrifuge tube (SW 27). Carefully lay on top of it 10mL of 3M CsCl. Load the phage suspension over 3M layer. Cellulose nitrate tube should be filled full Otherwise the tube will collapse during centrifugation.
 
     
 
 
     




     
 
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