Recovering Yeast 2-hybrid plasmids from yeast

Yeast plasmid Extraction Protocol Using Zymolyase and Manifold Miniprep Method

This is a simple and efficient way to recover plasmids from yeast. There are no glass beads and no phenol:chloroform steps.

 Solutions Requried:

67 mM KH2PO4  pH 7.5 (This is the resuspension solution used in Clontech YeastmakerTM)

25 ug/ul Zymolyase/Lyticase  (~0.5 units/ul) (Sigma #L2524  or amsbio  Zymolyase)  in 0.01M Na2HPO pH=7.5 in 50% glycerol (store at –20C). You can also use 10mM Tris pH=7.5 as the buffer.

Lysis sol’n (1ml 10%SDS, 200ul 10N NaOH in 10 ml of H2O)

Neutralizing sol’n (4M KoAc pH 5.5) Note:  The Lysis (aka Sol’n II)  and Neutralizing (aka Sol’n III) solutions are the same as the ones used in our bacteria miniprep solutions.

Silica Slurry and Wash Solution

 Procedure:

1.      Obtain 1ml of saturated yeast culture grown in selection media (i.e. –Leu) and spin down at full speed for 1 min., then aspirate the supernatant.

2.      Resuspend pellet in 50ul- 100ul of 67mM KH2PO4  pH 7.5 and vortex.

3.      Add 10ul Zymolyase (25 ug/ul (0.5  units/ul) in 0.01M Na2HPO4 and 50% glycerol) Incubate at 37°C for 1 hour. This step degrades the yeast cell wall creating spheroplasts and from this step onward we treat just like a plasmid mini prep for E. coli. Note:  other protocols use 10ul of 5 uint/ul zymolyase or up to 200 units. You may have to use a higher concentration if spheroplasts are not formed.

4.      Add 200ul Lysis Sol’n (1ml 10%SDS, 200ul 10N NaOH in 10 ml of H2O) and mix by inversion.

5.      Add 200ul Neutralizing Sol’n 4M KoAc pH 5.5) and mix by inversion.

6.      Spin at full speed for 10 mins.

7.      Add supernatant to manifold columns containing 200ul celite (silica) slurry and isolate DNA using our miniprep method (wash 2X with Wash Sol’n, spin to dry, and elute columns in new microfuge tube). Alternatively you can pass the supernatant over a Qiagen mini prep column. Elute in 50 to 100ul EB or TE.

 Use 5 to 10 ul of DNA to transform E. coli (i.e. MH6 -LEU)  competent cells and grow colonies on Amp plates. (note: If pGBKT7 (Kan R) was used for your bait vector then you can just transform into XL1 Blue competent cells and select for  Kan resistance.)

Patch single colonies onto M9 -LEU plates (if your library is –LEU). If colony grows overnight at 37°C then grow up colony in 2XTY + AMP overnight, and purify by usual bacteria Miniprep Method.

 

Silica Slurry:

Silica Suspension:

resuspend immediately before use by shaking vigorously

Wash Solution:

50 mM NaCl, 10 mM Tris 7.5, 2.5 mM EDTA, 50% Ethanol

Procedure:

  1. Prepare Silica Suspension
    • suspend 2-10  grams of Silica (Sigma S-5631) in 20 mls H2O and allow to settle for 2 hrs
    • remove milky supernatant and suspend settled silica in 20 mls H2O and allow to settle
    • repeat two more times
    • estimate volume and resuspend silica in 2 vols 6M Guanidine Hydrochloride-1M KOAc buffer, pH 5.5.