The following protocol is intented for providing a gentic confromation that a observed phenotype of a constructed deletion strain (using hygromycin resistance) is caused by the introduced mutation (essentially checking for unintented and unmarked secondary mutations in the strain). The cross of the mutant and the tester (here wild type) should show that the observed phenotype is 100% linked to the hygromycin resistance.
However, with small modifications the protocol can also be used for construction of double deletion strains by varying the selection pressure and assay conditions.
Incubate for an additional 10 – 14 days.
Transfer the perithecium to an Eppendorf tube with 400 µl sterile MilliQ water and squeeze the perithecium using the pair of forceps to release the ascospores from the perithecium.
Vortex briefly and transfer 20 µl spore suspension to a microscope slide to check the ascospore quality in a microscope.
Incubate at 20 oC for 3 days to allow spore germination.
Transfer all colonies from the 14 cm PDA agar plates to PDA and PDA hyg150 agar plates using s sterile toothpick. There is room for 34 colonies on each plate.
Incubate at 20 oC for 4 days.
PDA PDAhyg75 After 2 days
PDA PDAhyg75 After 5 days
Preparing the carrot-agar (Klittitch 1988)
Tween 60 solution, 2.5 %
Tween 60 stock is highly viscous; microwave about 20 seconds to liquefy. Using wide-bore pippet, transfer 3.75 ml to a 250 ml Pyrex bottle containing 146.5 ml warmed deionised water. Autoclave.
Dette sted blev sidst opdateret 14. July 2010