|Isolation of Shuttle Vectors from Yeast and Transformation into E. coli |
- Grow an overnight culture for ~1.5 - 2 days (until as turbid as a bacterial miniprep o/n)
- Spin 13000 rpm 5 min to pellet, and aspirate s/n
- Add 100 µl 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.
- Add 100 µl phenol/ CHCl3 plus ~60 ul of glass beads (Crown Corning Braun 0.45-0.5mm) i.e., 1/3 rd vol of soln.
- 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.
- Heat 65°C 5 min.
- Spin 13000 rpm 5 min, extract s/n (there's usually a lot of proteinatious material at the interphase). Repeat phenol/ CHCl3 extraction.
- 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.
- Mix by inverting or pipetting - dispense into 3 ml syringe which is attached to the column placed on the "Pig".
- Pull vacuum through to pack down resin.
- 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.
- Allow 2-3 min for residual wash buffer to run through resin - disconnect vacuum, remove syringe.
- Place column in an ependorf tube, spin at high speed for 1 min to remove residual wash buffer.
- 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.
- 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.
There are 3 main advantages in using this method (glassbeads/ purified DNA):
- yeast cells can be grown for a shorter time period < 2 days
- electrocompetent cells of lower efficiency can be used
- some yeast clones have very low yields of the GADGH vector DNA, therefore a higher amt of DNA may be electroporated.