To begin, inoculate a single OP50 colony in five milliliters of LB with ampicillin and amphotericin B and incubate for approximately six hours at 37 degrees Celsius in a bacterial shaker. Once the culture becomes cloudy, dilute the preinoculum culture of OP50 by 1 to 2, 000 in 250 milliliters of TB with ampicillin and glycerol. Then transfer the OP50 liquid culture into a sterile centrifugation tube.
Centrifuge the tube for 15 minutes at 3, 100 G in a tabletop centrifuge at four degrees Celsius. Discard the supernatant and resuspend the OP50 pellet in sterile water before recentrifugation. After the second wash, discard the supernatant and resuspend the OP50 pellet in sterile water to a volume of 50 milliliters.
Transfer it to a pre-weighed 50 milliliter conical tube. Centrifuge the tube for 20 minutes to pellet the OP50. At the end of the centrifugation, carefully remove all remaining supernatant, ensuring no water remains in the tube.
Calculate the weight of the pellet by subtracting the weight of the empty centrifugation tube from the weight of the centrifugation tube with the pellet. Thoroughly resuspend the OP50 pellet in S-complete buffer to achieve a concentration of 100 milligrams per milliliter. Test the OP50 suspension on an agar plate to check for any contamination.
Store the OP50 suspension in S-complete buffer at four degrees Celsius.
