酵母快速转化(Quick Yeast Transformation)
互联网
1976
When just a few transformants are sufficient, such as the transformation of a test plasmid or a GAL4BD fusion plasmid, the following protocol can be used. This procedure is very flexible and can be applied to yeast cells from a number of different sources; however, for best results, use cells from a freshly grown plate.
- Scrape a 25 µl yeast inoculum for EACH transformation planned from a plate with a sterile toothpick or loop and resuspend in 1 ml of sdd water. (Each Transformation reaction requires about a 25 µl volume of yeast cells, therefore if you scrape about 100 µl volume of yeast cells into 1 ml this represents 4 transformation reactions~undefined~1~0~0~0~0~0~0~Kp~MTIP~I~K~Hp~M~1~0~0~0~0~0~0~Kp~MThe_plate_can_be_up_to_a_week_old~E_yet~E_fresh_inocula_are_preferred._Yeast_cells_taken_from_most_types_of_media_plates_can_be_transformed_by_this_method~E_although_cells_grown_on_YPAD_perform_best._Remove_cells_from_the_edge_of_a_cell_mass_or_colony_to_ensure_recovering_healthy_cells~K~Hp~M~1~0~0~0~0~0~0~Kul~M~K~Hul~M~1~0~0~0~0~0~K~Hli~M~1~0~0~0~0~0~Kli~MPellet_the_cells_at_top_speed_in_a_microfuge_for_5_sec.~K~Hli~M~1~0~0~0~0~0~Kli~M~1~0~0~0~0~0~0Resuspend_the_cell_pellet_in_1_ml_of_100_mM_LiAc_and_incubate_for_5_min_at_30~1~0~0~0~0~0~0~Ksup~Mo~K~Hsup~M~1~0~0~0~0~0~0C.~1~0~0~0~0~0~K~Hli~M~1~0~0~0~0~0~Kli~MPlace_the_volume_representing_a_single_TRANSFORMATION_REACTION_~Aor_25_µl volume of yeast cells, If you scraped a 50 µl volume of yeast cells into 1 ml of sterile water then this volume would be 500 µl ) into a separate microcentrifuge tube and spin the suspension at top speed in a microcentrifuge for 5 sec. Remove the supernatant with a micropipet. .
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Add the following components into the tube on top of the cell pellet in this order or as a premix;.
Premix (µl) 1X 3X 5X Peg (50% w/v) 240 720 1200 LiAc (1.0 M) 36 108 180 SS-DNA (10mg/ml) 10 30 50 H 2 O 64 192 320
Use 350 µl per tranformation
5.0 µl of plasmid DNA (100 ng to 5 µg) - Vortex the cell pellet for at least 1 min to resuspend the cell pellet in the transformation mix and incubate at 42 o C for 20 min.
- Pellet the cells at top speed in a microcentrifuge for 10 sec. Remove the supernatant using a micropipet.
- Gently resuspend the pellet in 200 - 400 µl of sdd water by slowly pipetting up and down.
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Plate the cell suspension onto 1 or 2 plates of SC ommision medium that selects for the presence of the plasmid. Colonies should be visible in 2 - 4 days at 30
o
C.
Yield will range from a few hundred to a few thousand transformants.
