| - suspend a single plaque in 1 ml PSB
- adsorb 10 min at 37°C: 0.1 ml eluted phage*/0.1 ml MgCa/0.1 ml saturated K802 culture grown in NZY broth/0.2% maltose
- use 10 ml of this to inoculate 2.5 ml NZY, shake ovn at 37°C or until lysed
- cool to RT, then add 2 drops of CHCl3
- add 5 ml of 10 mg/ml DNase I (20 mg/ml final) and gently shake for 25 min
- divide lysate into two Eppendorf tubes, spin 5 min
- transfer 0.6 ml of each tube into a new tube prefilled with 200 ml of STE
- mix and incubate 15 min at 70°C
- cool to RT, add 150 ml 8 M KAc, mix and keep on ice for 15 min, spin for 15 min
- save 700 ml of clear supernatant, extract 1x with phenole/chloroform (vortex 5 sec)
- add 420 ml 2-propanol, mix and let sit at RT for 10 min
- spin for 8 min, very carefully remove supernatant (pellet does not stick to bottom of tube very well)
- wash pellet twice with 0.5 ml of 70% EtOH (-20°C), dry in speed vac
- digest pellet in 50 ml each of 0.3 mg/ml panc. RNase in TE, pH 8.0 for 30 min at 37°C
- pool to 100 ml per sample, add 40 ml of 5 M NH4Ac and 200 ml 2-propanol
- keep 10 min at RT, spin 10 min
- remove supernatant, wash with 0.5 ml of 70% EtOH (-20°C), dry in speed vac
- take up pellet in 20 ml 1x TE, usually 10 ml can be used for a restriction digest
Solutions: Remarks: *: this value holds true for genomic phages (EMBL4, Lambda DASH), if using cDNA phages (lgt10/11, Lambda ZAP) take: 1 ml of eluted phage in each of 0.1 ml l-dil, MgCa and K802. This is a tricky protocol. However, when strictly adhering to the indicated treatment times and generally experimenting carefully it should work allright. |