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
+210 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 MgCl
2.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 MgCl
2use 9-10 ml per large plate
SM
5.8 g NaCl
2.0 g MgSO
4 . 7H2O50 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.
