LambdaPhageDNAQuickprep

1. suspend a single plaque in 1 ml PSB

2. 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

3. use 10 ml of this to inoculate 2.5 ml NZY, shake ovn at 37°C or until lysed

4. cool to RT, then add 2 drops of CHCl₃

5. add 5 ml of 10 mg/ml DNase I (20 mg/ml final) and gently shake for 25 min

6. divide lysate into two Eppendorf tubes, spin 5 min

7. transfer 0.6 ml of each tube into a new tube prefilled with 200 ml of STE

8. mix and incubate 15 min at 70°C

9. cool to RT, add 150 ml 8 M KAc, mix and keep on ice for 15 min, spin for 15 min

10. save 700 ml of clear supernatant, extract 1x with phenole/chloroform (vortex 5 sec)

11. add 420 ml 2-propanol, mix and let sit at RT for 10 min

12. spin for 8 min, very carefully remove supernatant (pellet does not stick to bottom of tube very well)

13. wash pellet twice with 0.5 ml of 70% EtOH (-20°C), dry in speed vac

14. digest pellet in 50 ml each of 0.3 mg/ml panc. RNase in TE, pH 8.0 for 30 min at 37°C

15. pool to 100 ml per sample, add 40 ml of 5 M NH₄Ac and 200 ml 2-propanol

16. keep 10 min at RT, spin 10 min

17. remove supernatant, wash with 0.5 ml of 70% EtOH (-20°C), dry in speed vac

18. take up pellet in 20 ml 1x TE, usually 10 ml can be used for a restriction digest

Solutions:

STE: 1.5 % SDS, 0.3 M Tris (pH 9), 0.15 M EDTA STE 10 % SDS7.5 ml2 M Tris pH 97.5 ml0.5 M EDTA15.0 mlTotal50.0 ml		l-dil: 10 mM Tris-HCl pH7.5/10 mM MgSO4, autoclave before use l-dil 2 M Tris-HCl pH 7.55.0 ml1 M MgSO410.0 mlH2O985.0 mlTotal1 l
MgCa: 10 mM MgCl2/10 mM CaCl2, autoclave before use MgCa 1 M MgCl25.0 ml1 M CaCl25.0 mlH2O990.0 mlTotal1 l		PSB: 10 mM Tris-HCl pH 7.5, 0.1 M NaCl, 10 mM MgCl2, 0.05 % gelatine; store over CHCl3 PSB 2 M Tris-HCl pH 7.51.0 ml5 M NaCl4.0 ml1 M MgCl22.0 ml0.5 % gelatine20.0 mlH20173.0 mlTotal200.0 ml

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.