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RESEARCH DIVISION Laboratory Manual

 


 

Isolation of Shuttle Vectors from Yeast and Transformation into E. coli

  1. Grow an overnight culture for ~1.5 - 2 days (until as turbid as a bacterial miniprep o/n)
  2. Spin 13000 rpm 5 min to pellet, and aspirate s/n
  3. Add 100 l lysis buffer (2.5 M LiCl, 50 mM Tris pH 8.0, 20 mM EDTA pH 8.0, 4 % Triton-X 100), vortex to resuspend.
  4. Add 100 l phenol/ CHCl3 plus ~60 ul of glass beads (Crown Corning Braun 0.45-0.5mm) i.e., 1/3 rd vol of soln.
  5. Vortex tubes vigorously by hand, initially, for 3 min - you can do 4-5 tubes simultaneously; then place all tubes into a red rack and strap onto vortexer for 10 min at high speed.
  6. Heat 65C 5 min.
  7. Spin 13000 rpm 5 min, extract s/n (there's usually a lot of proteinatious material at the interphase). Repeat phenol/ CHCl3 extraction.
  8. To the s/n add 0.5 ml of Wizard miniprep resin (Promega) - the Wizard protocol calls for 1 ml of resin, but since the vol of s/n is only ~ 100 ul, 0.5ml should be sufficient to maintain the ionic interaction.
  9. Mix by inverting or pipetting - dispense into 3 ml syringe which is attached to the column placed on the "Pig".
  10. Pull vacuum through to pack down resin.
  11. Wash 3X with 1 ml column wash buffer (55 % Ethanol, 200 mM NaCl, 20 mM Tris pH 7.5, 5 mM EDTA) - the conc of salt, tris and EDTA is twice that of the Wizard protocol but it still works and I've stuck with these conc's.
  12. Allow 2-3 min for residual wash buffer to run through resin - disconnect vacuum, remove syringe.
  13. Place column in an ependorf tube, spin at high speed for 1 min to remove residual wash buffer.
  14. Elute DNA off resin with 50 ul TE or dH2O into a clean eppe - add TE, stand 1 min then spin high speed 1 min.
  15. Use 2 ul to transform electrocompetent cells (with transformation efficiency of at least 5 X 107 colonies/ ug) - plate 50-150 ul of 1ml onto small petri dish.

NOTES :

There are 3 main advantages in using this method (glassbeads/ purified DNA):

  1. yeast cells can be grown for a shorter time period < 2 days
  2. electrocompetent cells of lower efficiency can be used
  3. some yeast clones have very low yields of the GADGH vector DNA, therefore a higher amt of DNA may be electroporated.
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998