Plasmid Rescue from Yeast cells

Adapted from the Gottschling Lab who obtained it from Trey Powers (Fields Lab),  also reported by Michael Jones  (using the Macherey-Nagel NucleoSpin Plasmid kit)

 

1. 1. Grow yeast overnight.
   2. Spin down 1.5 mL to 4 mL of culture 5 minutes at 4600 rpm.
   3. Decant.
   4. Add 250 µL of buffer A1, transfer into a microfuge tube and add about 250 µL of glass beads to each tube.
   5. Beat or vortex on high for 3-5 minutes.
   6. Add 250 µL of buffer A2 and gently mix well.
   7. Incubate at room temperature for up to 5 minutes. (Do not let lysis reaction continue for more than five minutes).
   8. Add 300 µL of buffer A3 and mix immediately, but gently.
   9. Centrifuge for 5-10 minutes on full blast.
   10. Load supernatants (max 750uL) to NucleoSpin Plasmid column.
   11. Centrifuge column for 1 minute on full blast. Discard flow-though.
   12. Wash column with 500 µL of Buffer AW and centrifuge 1 minute. Discard flow-though.
   13. Wash column with 600 µL of Buffer A4 and centrifuge 1 minute.
   14. Discard flow-through and spin for 2 additional minutes.
   15. Place column in a clean tube, add 10 µL of buffer AE, then add 50 uL of water.
   16. Let sit for 1 min. then spin for 1 min.
   17. For efficent transformation into coli use 3 µL of this prep per 50 uL aliquot of E. coli (high efficency) using the 30 sec heat shock protocol. (NOTE: using up to 20 µl will increase yield of transformants)

Dernière mise à jour : ( 18-03-2019 )