RonDavis:YeastTransformation (Uli)

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Ulrich Schlecht (Talk | contribs)
(Yeast transformation using Lithium Acetate)
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Revision as of 19:27, 7 October 2013

1 inoculate an overnight culture (5 ml) and grow to saturation 2 inoculate 1 ml of the saturated culture into 10 ml of fresh YPD and grow for 2 to 6 hours 3 transfer 1 ml into a 1.5 ml Eppendorf tube (this is enough for 4 transformations) 4 spin for 2 min at 8000 rpm 5 wash with 1 ml of 100 mM LiAc 6 spin for 2 min at 8000 rpm 7 wash with 1 ml of 100 mM LiAc 8 resuspend in 200 ul Salmon Sperm DNA (boil for 10 min and then incubate on ice prior to use) 9 aliquot 20 ul of cell suspension into a 1.5 ml Eppendorf tube 10 add DNA to be transformed (PCR product, plasmid, or vector + insert) 11 incubate for 30 min at 30 degrees C 12 resuspend the pellet in 270 ul PEG (48%) 13 add 30 ul of 1 M LiAc 14 incubate for 30 min at 30 degrees C 15 heat-shock for 30 - 60 min in water bath at 42 degrees C 16 spin for 30 sec at 14000 rpm 17 remove supernatant 18a if selecting for a dominant drug-marker(such as KanMX) resuspend the pellet in 5 ml of YPD and incubate overnight at room temperature 18b if selecting for an auxotrophic marker (such as URA3, LEU2, HIS3) no recovery in YPD is needed 19 plate onto selective media and incubate at 30 degrees C 20 colonies should appear after ~ 2 days

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