M13KO7 HELPER PHAGE
STORE: +4C, DO NOT FREEZE
SHIP: Wet Ice Only
M13K07 HELPER PHAGE PROPAGATION
It is important that the M13KO7 Helper Phage be grown from fresh plagues.The following procedure produces an inoculum of M13KO7 Phage that remain viable for more than 1 year when stored at 4C.
1.Streak M13K07 on a l×YT agar plate.
2.Pour 4 ml of soft agar containing 0.5 ml of a culture of NM522 >0.8OD660）across the plate from the dilute side of the streak toward the more concentrated side.
3.Incubate 6-12 hours at 37C.Scrape areas of closely spaced single plaques into 30-200 ml of 2×YT Media containing 70 ug/ml Kanamycin.
4.Grow at 37C for 10-14 hours with very good aeration. Pellet the cells, repeat centrifugation until a cell pellet is no longer produced. Remove the supernatant. The supernatant will serve as inoculum of M13KO7 for more than 1 year if stored at 4C.The titer of M13KO7 produced should be>5x
PRODUCTION OF SINGLE STRAND
1. Grow phagemid transformed cells to Asw-0.5-0.8 density (1 A660=8×108 cells)at 37C in 2×YT media supplemented with 0.001% thiamine and 150 ug/ml ampicillin.
2. Infect 2ml of this culture with M13KO7 at a multiplicity of infection (m.o.i) of 10.Shake at 300 rpm for 1 hour at 37C in a 50 ml tube.
3. After 1 hour, mix 400 ul of infected cells with 10 mls of 2×TY media Add kanamycin to 70 ug/ml and grow at 37C for 14-18 hours with 300 rpm agitation.
4. Centrifuge to remove cells. Repeat until a pellet is no longer produced. supernatant contains the single-stranded "phage".
5. Add 0.25 volume of a 3.5 M ammonium acetate/20% polyethylene glycol solution. Mix by inversion and place on ice for 30 minutes. Collect precipitated phage by centrifugation for 15-30 minutes at 11,000×g. Carefully remove supernatant. Allow the tubes to drain and then remove any excess liquid by aspiration.
6. Resuspend the pellet in 400 μl TE buffer, with gentle vortexing. Perform subsequent steps in microcentrifuge tubes.
7. Add an equal volume of phenol/chloroform and vortex for 30 seconds. Centrifuge for 1 minute to separate the phases. Carefully remove the upper aqueous layer and transfer it to a clean tube. Re-extract the aqueous phase
8. Extract with an equal volume of chloroform, separate the phases by centrifugation, and transfer the upper aqueous phase to a clean tube. Re-extract the aqueous phase.
9. Add 0.1 volume of 3 M sodium acetate (pH 5.0) and two volumes of 100% ethanol, mix and place on dry ice for 15 minutes. Pellet the DNA in a microcentrifuge. Remove the supernatant and discard. Wash the pellet with 1ml of ice-cold 70% ethanol. Carefully remove the supernatant and briefly dry the pellet under vacuum.
10. Dissolve the DNA pellet in 20 ul of Tris buffer and store at -20℃.The A260/A280 ratio of the solution should be at least 1.7 for DNA sequencing.
Safety warnings and precautions
This product should be handled only by persons trained in laboratory techniques and used in accordance with the principles of good laboratory practice. All chemicals should be considered potentially hazardous; therefore, when handling chemical reagents, it is advisable that suitable protective clothing, such as laboratory overalls, safety glasses and gloves be worn. Care should be taken to avoid contact with skin or eyes. In case of contact with skin or eyes, wash immediately with water.
Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.