IGEM:Virginia 2012/Protocols/CaCl2 Competent Stock Cells
This is how to isolate the phage genome from a preparation of bacteriophages, specifically the Bordetella phage.
- 10 mM EDTA
- CsCl-banded phage
- sterile tube
- 100% ethanol
- -20°C freezer
- 0.5 mL of 70% ethanol
- TE Buffer
- 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)
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- Who has experience with this protocol?
or instead, discuss this protocol.