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Worm Breeder's Gazette 17(2): 22 (April 1, 2002)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Laboratory of Molecular Neurobiology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan
In reverse genetics using transposon or chemical mutagen, recovery from freezing stock is one of the important steps to isolate deletion mutants. Effective isolation of deletion mutants requires a "good" library, which has a high survival rate as same as a high mutation rate. Although mutagenesis and PCR conditions have been optimized previously, the effect of freezing condition on the survival rate has not been fully investigated. Here, we report that the volume of freezing solution affects survival rate. Particularly, the survival rate dramatically decreased in small volume.
N2 Bristol strain was used. Three or four adult worms were placed on a seeded 60 mm NGM plate and cultured at 20 °C for 5 days until bacterial lawn became almost invisible. Eight or ten plates were used for experiment 1 or experiment 2. Freshly starved L1 larvae (and adults) were washed from the plates with M9 buffer into a 15 ml plastic tube. After low speed centrifugation, the supernatant was removed and an equal volume of 2 x freezing solution was added. The aliquot (worm solution) was mixed gently and transferred into 2 ml freezing vials. In experiment 1, each set of vials contains various volume of worm solution, but all vials contain about 50,000 worms. In experiment 2, each set of vials contains various volume of worm solution, in which a density of worms was about 50 worms/µl. The vials were placed in holes drilled into a styrofoam block without a lid. The block was placed in the -80 °C freezer for overnight. In the next day, vials were thawed and the contents of each tube were plated onto a seeded 60 mm NGM plate. Two days later, the number of surviving worms on each plate was counted and the survival rate was determined (average±SD, n=3).
Small volume (100 µl) of worm solution showed the lowest survival rate (Experiment 1). The survival rate of 100 µl was 4 times lower than those of 500 µl or 2000 µl. In experiment 1, volume of worm solution and density of worms were both changed. In experiment 2, survival rate were determined in various volumes with the same worm density to determine which factor affects survival rate. Small volume (100 µ) also showed the lowest survival rate, and 500 µl of volume showed a relatively high survival rate but large SD (Experiment 2). Over 1000 µl of volume resulted in stable and high survival rates. A relatively low density of worms seems to give a better survival rate.
Standard freezing methods recommend that the rate of temperature decrease should be slow (about 1 °C/min). We assume that the rate of temperature decrease depends on the volume of worm solution and that the temperature in small volume probably decrease rapidly. On construction of freezing library of mutagenized worms, 200 µl PCR tubes or 96-well plates were often used to save the space in a freezer. However, our results showed that small volume of freezing worm solution would result in a large decrease of survival rate. We suggest that the rate of temperature decrease should be controlled carefully, in order to achieve better efficiency for isolating deletion mutants from the frozen library with small volume.
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