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D.3

Protocol D.3

Lambda DNA Preparation

This is a plate method that gives very good yield for cloning. I have combined the Promega and Maniatis protocols.

Solutions

 

T-TYN Media + Mg+2

10 g tryptone

5 g yeast extract

5 g NaCl

10 ml 1M Tris pH 7.2

up to 1 liter with Q, adjust pH to 7.2 with NaOH.

Autoclave, cool and add 10 ml 1M MgCl2.

NOTE: FOR GROWING UP CULTURES OF Y1090r- ADD 1 ml 20% MALTOSE PER 100 ml.

 

T-TYN Plates

make 1 liter T-TYN media

add 15 g agar

autoclave, cool and add 10 ml 1M MgCl2

pour 10 large plates

 

T-TYN Top Agarose

make 1 liter T-TYN media

add 7 g ultrapure agarose

autoclave, cool and add 10 ml 1M MgCl2

use 9-10 ml per large plate

 

 

 

 

 

SM

5.8 g NaCl

2.0 g MgSO4 . 7H2O

50 ml 1M Tris 7.5

5 ml 2% gelatin

up to 1 liter with Q

autoclave and store at room temperature

 

Procedure

• Innoculate an overnight culture with Y1090r- in T-TYN + Mg+2 + maltose + ampicillin. Dilute 2 ml of this overnight with 3 ml media and infect 500 ml with 50,000 pfu. Shake for 20-30 minutes at 37° and plate onto 150 mm plates with 10ml T-TYN top agarose.

• Incubate overnight at 37° and add 10 ml SM. Place on tiltboard at 4° for 2-3 hours and remove SM. Add an additional 5 ml of SM and drain 5 minutes. Add 5 ml chloroform to the phage stock and remove supernatant.

• Add DNaseI and RNaseI to a final concentration of 1 mg/ml and incubate at room temperature for 30 minutes.

• Add 2.9 g NaCl per 50 ml of phage supernatant and incubate on ice for 1 hour. Spin at 11,000 x g for 10 minutes to remove the debris.

• Add 5 g PEG 8000 per 50 ml of phage supernatant and hold on ice for 30 minutes.

• Spin at 11,000 x g for 10 minutes to pellet the bacteriophage. Drain and resuspend in 10 ml SM. Add 10 ml chloroform, and spin at 3,000 x g for 15 minutes. Remove the supernatant and phenol/chloroform extract twice.

• Add 1 volume of isopropanol and incubate at -80° C for 1 hour. Spin at 11,000 x g for 10 minutes. Wash with 80% EtOH and dry. Resuspend the pellet in 200 ml Q. Use RNaseA in restriction digests.