Quick Shotgun Cloning of Phage Inserts
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digest genomic phage DNA:
EcoRI Digestion 10x EcoRI buffer 0.5 ml phage DNA (200 ng) 4.5 ml Eco RI (20 u/ml) 0.2 ml total 5.2 ml
- incubate for 15 min at 37°C (heating block or water bath)
- heat for 5 min at 65°C (heating block)
- add 2 ml 5 M NH4 Ac and 10 ml isopropanol
- spin for 20 min at 4°C
- wash pellet with 70% EtOH (-20°C), speed vac and dissolve pellet in 4 ml 0.1 x TE (ca 50 ng/ml)
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prepare a ligation mix:
Ligation Mix (2x) 10x ligase buffer 1.0 ml digested vector
(0.1 mg/ml)1.0 ml H2 O 6.0 ml total 8.0 ml
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divide ligation mix between two Eppendorf tubes
Ligation Rxn Insert Control ligation mix 4.0 ml 4.0 ml insert 1.0 ml --- ml T4 DNA ligase (400 u/ml) 0.2 ml 0.2 ml Total 5.2 ml 4.2 ml - incubate for 2-3 h (or ovn) at 14°C
- proceed with the transformation of the appropriate E. coli strain
Solutions:
10x Ligation Buffer:
0.5 M Tris-HCl pH 7.8, 50 mM MgCl2 , 0.1 M b-mercaptoethanol, 50 mM ATP, 5 mg/ml BSA
Ligation Buffer (10x) 1 M Tris-HCl pH 7.8 500.0 ml 1 M MgCl2 50.0 ml b-mercaptoethanol 7.0 ml 100 mM ATP 50.0 ml 0.1 g/ml BSA 50.0 ml H2 O 343.0 ml Total 1 ml
Remarks:
Quickprep phage DNA gets prepared according to our standard protocol . We use Lambda DASH or EMBL4 lambda phages as vectors. Thus the insert gets released using EcoRI. Be aware that the insert usually carries internal EcoRI sites as well.
Materials:
Reagent/Tool | Supplier | Cat.-# |
BSA | ||
ATP | ||
T4 DNA Ligase | ||