CGSC FAQ on Procedures
Here are some Frequently Asked Questions on procedures that we use and other widely used genetic procedures.
1. What procedure do we use to lyophilize strains?
There are many protocols for this. We use the following:
0.1 ml of a glycerolized, or non-glycerolized culture is distributed on the surface of a slant and incubated overnight at 30 C. (We use 30 C, because most ts as well as non-ts cultures can be grown in this incubator.) Small rectangular strips, appropriate to size of lyophil tube, of Whatman #3 are cut and the date and identification information for the strain are pencilled onto the strips (mixing up id's being the largest sin we can commit). The paper is creased longitudinally and placed in the tube, touching bottom. The open tube is covered with a "glass lyophil tube cover" (i.e., whatever your setup uses for tubes and covers), and the racks of tubes and filters are autoclaved. The culture on the slant is overlaid with .5 ml. 10% glucose and .5 ml. 10% peptone and the cells suspended. The suspended cells are transferred via sterile pasteur pipette to the sterilized filter paper in the lyophil tube until the paper is moistened to "almost saturation". The tubes are placed on the lyophil ports.
2. What procedure do we use for mailing cultures on sterile disks?
We use either a glycerolized culture frozen at -70 or -80C, or less frequently a glycerolized culture at -20C (which is a non-frozen slurry that can be pipetted without thawing and refreezing, used only for shorter-term, heavily used cultures). For the non-frozen culture we spot 0.1 ml. onto the small filter paper disk, which has been autoclaved on a square of saranwrap, both sandwiched between large filter disks, the sandwiches stacked in a glass petri dish and after inoculation, wrap the disk carefully and tightly in the underlying saran wrap. For the deep-frozen culture, we take a "chunk" from the frozen glycerol culture and place that on the disk and add 0.1 ml. Luria broth to moisten the filter, and wrap as above. We want enough liquid to make sure that the filter is entirely moistened, not partially dry. The filter paper disks we use are Schleicher and Schuell (S&S) #740-E, 1/2 in. circles, absorbent disks suitable for antibiotic disk assays. Probably any pure, absorbent filter paper would do. For our -70 C. cultures, we use a final concentration of 15% glycerol; for an occasional temporary working culture that we want to keep at -20 C without freezing and thawing for sampling, we use 25% glycerol.
3. What is the procedure for P1 transduction?
Here is a link to a protocol on OpenWetWare from Robert Sauer's lab at MIT. Sauer: P1vir Phage Transduction
This is the link for other protocols on OpenWetWare.