IGEM:Virginia 2012/Protocols/Isolating the Phage Genome

Overview

This is how to isolate the phage genome from a preparation of bacteriophages, specifically the Bordetella phage.

Materials

• 10 mM EDTA
• CsCl-banded phage
• sterile tube
• 100% ethanol
• -20°C freezer
• Centrifuge
• 0.5 mL of 70% ethanol
• TE Buffer

Procedure

• Add 10 volumes of 10mM EDTA pH 8.0 to 1 volume of CsCl-banded phage in a sterile tube
• Heat for 5 minutes at 65°C; let cool to room temperature
• Add 2.5 volumes 100% ethanol; place in -20°C freezer for 20 minutes
• Centrifuge: 10,000g for 5 minutes
• Discard supernatant; rinse pellet with 0.5ml 70% ethanol; centrifuge again; discard supernatant; rinse pellet with 0.5ml 70% ethanol; centrifuge again; discard supernatant
• Let pellet air dry at room temperature
• Resuspend DNA in TE buffer; let DNA sit for several hours to go into solution; or, restriction digests can be performed immediately; store solution of DNA at -20°C for long-term storage (i.e. months) or at 4°C for short-term storage (i.e. 1-2 days)

Notes

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