λ噬菌体的DNA的快速制备
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1900
1. suspend a single plaque in 1 ml PSB
2. adsorb 10 min at 37°C: 0.1 ml eluted phagundefined/0.1 ml MgCa/0.1 ml saturated K802 culture grown in NZY broth/0.2% maltoseuse 10 ml of this to inoculate 2.5 ml NZY, shake ovn at 37°C or until lysed
3. cool to RT, then add 2 drops of CHCl3
4. add 5 ml of 10 mg/ml DNase I (20 mg/ml final) and gently shake for 25 min
5. divide lysate into two Eppendorf tubes, spin 5 min
6. transfer 0.6 ml of each tube into a new tube prefilled with 200 ml of STE
7. mix and incubate 15 min at 70°C
8. cool to RT, add 150 ml 8 M KAc, mix and keep on ice for 15 min, spin for 15 min
9. save 700 ml of clear supernatant, extract 1x with phenole/chloroform (vortex 5 sec)
10. add 420 ml 2-propanol, mix and let sit at RT for 10 min
11. spin for 8 min, very carefully remove supernatant (pellet does not stick to bottom of tube very well)
12. wash pellet twice with 0.5 ml of 70% EtOH (-20°C), dry in speed vac
13. digest pellet in 50 ml each of 0.3 mg/ml panc. RNase in TE, pH 8.0 for 30 min at 37°C
14. pool to 100 ml per sample, add 40 ml of 5 M NH4Ac and 200 ml 2-propanol
15. keep 10 min at RT, spin 10 min
16. remove supernatant, wash with 0.5 ml of 70% EtOH (-20°C), dry in speed vac
17. take up pellet in 20 ml 1x TE, usually 10 ml can be used for a restriction digest
Solutions:
1. STE: 1.5 % SDS, 0.3 M Tris (pH 9), 0.15 M EDTA.
10 % SDS 7.5 ml; 2 M Tris pH 9 7.5 ml; 0.5 M EDTA 15.0 ml; Total 50.0 ml.
2. l-dil: 10 mM Tris-HCl pH7.5/10 mM MgSO4, autoclave before use.
2 M Tris-HCl pH 7.5 5.0 ml; 1 M MgSO4 10.0 ml; H2O 985.0 ml; Total 1 L.
3. MgCa: 10 mM MgCl2/10 mM CaCl2, autoclave before use.
1 M MgCl2 5.0 ml; 1 M CaCl2 5.0 ml; H2O 990.0 ml; Total 1 L.
4. PSB: 10 mM Tris-HCl pH 7.5, 0.1 M NaCl, 10 mM MgCl2, 0.05 % gelatine; store over CHCl3.
2 M Tris-HCl pH 7.5 1.0 ml; 5 M NaCl 4.0 ml; 1 M MgCl2 2.0 ml; 0.5 % gelatine 20.0 ml; H2O 173.0 ml; Total 200.0 ml.
Remarks:
~undefined: 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.
2. adsorb 10 min at 37°C: 0.1 ml eluted phagundefined/0.1 ml MgCa/0.1 ml saturated K802 culture grown in NZY broth/0.2% maltoseuse 10 ml of this to inoculate 2.5 ml NZY, shake ovn at 37°C or until lysed
3. cool to RT, then add 2 drops of CHCl3
4. add 5 ml of 10 mg/ml DNase I (20 mg/ml final) and gently shake for 25 min
5. divide lysate into two Eppendorf tubes, spin 5 min
6. transfer 0.6 ml of each tube into a new tube prefilled with 200 ml of STE
7. mix and incubate 15 min at 70°C
8. cool to RT, add 150 ml 8 M KAc, mix and keep on ice for 15 min, spin for 15 min
9. save 700 ml of clear supernatant, extract 1x with phenole/chloroform (vortex 5 sec)
10. add 420 ml 2-propanol, mix and let sit at RT for 10 min
11. spin for 8 min, very carefully remove supernatant (pellet does not stick to bottom of tube very well)
12. wash pellet twice with 0.5 ml of 70% EtOH (-20°C), dry in speed vac
13. digest pellet in 50 ml each of 0.3 mg/ml panc. RNase in TE, pH 8.0 for 30 min at 37°C
14. pool to 100 ml per sample, add 40 ml of 5 M NH4Ac and 200 ml 2-propanol
15. keep 10 min at RT, spin 10 min
16. remove supernatant, wash with 0.5 ml of 70% EtOH (-20°C), dry in speed vac
17. take up pellet in 20 ml 1x TE, usually 10 ml can be used for a restriction digest
Solutions:
1. STE: 1.5 % SDS, 0.3 M Tris (pH 9), 0.15 M EDTA.
10 % SDS 7.5 ml; 2 M Tris pH 9 7.5 ml; 0.5 M EDTA 15.0 ml; Total 50.0 ml.
2. l-dil: 10 mM Tris-HCl pH7.5/10 mM MgSO4, autoclave before use.
2 M Tris-HCl pH 7.5 5.0 ml; 1 M MgSO4 10.0 ml; H2O 985.0 ml; Total 1 L.
3. MgCa: 10 mM MgCl2/10 mM CaCl2, autoclave before use.
1 M MgCl2 5.0 ml; 1 M CaCl2 5.0 ml; H2O 990.0 ml; Total 1 L.
4. PSB: 10 mM Tris-HCl pH 7.5, 0.1 M NaCl, 10 mM MgCl2, 0.05 % gelatine; store over CHCl3.
2 M Tris-HCl pH 7.5 1.0 ml; 5 M NaCl 4.0 ml; 1 M MgCl2 2.0 ml; 0.5 % gelatine 20.0 ml; H2O 173.0 ml; Total 200.0 ml.
Remarks:
~undefined: 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.
