Melissa Cobb

LAMBDASORB LIQUID PHAGE PREP


1) Inoculate 200µl cells in a 50 ml sterile tube with either 2 plaques or 2 million pfus. Incubate for 20 minutes at room temp to allow adsorption. Add 15 mls of L-Broth containing 10mM MgSO4 and shake at 37°C until lysis (5-7 hrs). Add 150µl chloroform and shake an additional 15 min to lyse the remaining cells.
lambdasorb;
2) Spin at 3,000 for 5 minutes to pellet the cell debris and transfer the supernatant into a clean tube. Set aside 100µl as high titer stock. Add 100µl of LambdaSorb place tube on a rotator for 30 minutes at room temp or leave at 4°C overnight and rotate briefly in the morning.

3) Spin at 3,000 for 5 minutes. Discard supernatant and drain the tubes well.

4) Resuspend pellet in 1ml TM10 and transfer to eppendorf tube.

5) Spin very briefly (30 sec), discard supernatant and resuspend pellet in 500µl TE.

6) Add 5µl 10%SDS, 10µl 250mM EDTA and incubate at 65-70°C for 10 minutes.

7) Spin 1 minute to pellet protein debris and transfer the supernatant to a new tube. Add 50µg/ml of Proteinase K and incubate for 30 minutes at 37°C.

8) Phenol-Chloroform extract twice. Add 50µl 3M NaOAc and 500µl isopropanol and mix well. A DNA precipitate should form readily but, if not leave at -70°C for 15 min, thaw and centrifuge. If the precipitate is readily visible, centrifuge for only a minute so it won't be so hard to resuspend, otherwise spin for 10 minutes.

9) Resuspend DNA pellet in ~50µl TE.

NOTE: Some members of the lab find steps 7 and 8 to be unnecessary.