ssPhage DNA SPIN^TM Kit Protocol
Revision No. 2065-999-5F01W

The ssPhage DNA SPIN^TM Kit is designed to facilitate the isolation of single-stranded DNA from filamentous phage such as M13 or phagemids. Multiple samples can be processed in less than one hour without organic extractions, yielding pure ssDNA suitable for DNA sequencing and transformations. The ssPhage DNA SPIN^TM Kit contains sufficient reagents and materials to purify ssDNA from 60 or 120 phage cultures, depending on kit size. Reagents and materials supplied by the user (*) are 95% ethanol, sterile water or TE buffer, 1.5 ml microcentrifuge vials, and a microcentrifuge. The reagents and materials provided with the kit are for research purposes only.

Stab a single, well-isolated plaque with a sterile toothpick or needle and inoculate 2 ml of YT-broth (8g tryptone, 5g yeast extract, 5g NaCl/liter, catalog #3011-011) in a 5 - 10 ml sterile culture tube. Incubate the tubes with aeration at 37°C for 12-18 hours.

1. Centrifuge 1.5 ml of each culture in a microcentrifuge at full speed for 1 minute to pellet cells. Transfer 1.2 ml of each cell-free culture supernatant to a 1.5 ml microcentrifuge tube. (Optional: Filter the supernatant through a 0.45µ filter to remove any remaining cells and thus eliminate possible traces of cellular DNA or RNA in the final ssDNA elution).


2. To precipitate phage from the supernatant, add 350 µl of ssPhage Drop Buffer. Mix well and incubate 5-20 minutes (the longer times will increase yield 10-20%) at room temperature without agitation.


3. Centrifuge 5 minutes at maximum speed to pellet the phage. Completely remove the ssPhage Drop Buffer. Be careful not to lose the small visible phage pellet.


4. Resuspend the pellet in 10 µl TE or water; add 250 µl ssPhage GLASSMILK Spinbuffer (mix well before use; mix often during use to keep ssGLASSMILK in suspension). Incubate at room temperature 10 minutes with constant low speed vortexing to lyse the phage.


5. Centrifuge for 30 seconds at maximum speed and remove the supernatant. Resuspend the ssDNA/GLASSMILK pellet in 350 µl Wash Solution (add ethanol before use as instructed on the bottle). Transfer to a SPIN Filter and centrifuge 30 seconds at maximum speed to completely wash the DNA.


6. Elution: Transfer the filter to a clean 1.5 ml catch tube. Add 50 µl sterile water or TE to the Filter and centrifuge for 30 seconds at maximum speed. Remove and discard SPIN Filter. The ss phage DNA is now purified and ready for use.