Shauns Miniprep Protocol

 

Reminder ­ Wear Gloves, lab coat and eye protection!!!

 

1.      Vortex culture briefly and pour ~1.5mL into eppendorf tube.

2.      Spin @ max for 1 minutes.

3.      Aspirate media ­ be careful not to suck up pellet.

4.      Resuspend pellet in 250uL of Solution 1

5.      Add 250uL of Solution 2.  Cap and invert to mix ~ 8 times.

6.      Add 250uL of Solution 3.  Cap and invert to mix ~ 8 times.

7.      Spin @ max for 15 minutes.

8.      Transfer supernatant to new tube.  (~750uL)

9.      Add 750uL 100% Isopropanol.  Vortex and spin for 1 min @ max

10.   Aspirate supernatant.

11.   Wash w/ 750uL 70% EtOH.  Vortex briefly.

12.   Spin @ max for 1 min.

13.   Aspirate supernatant.

14.   Uncap and allow tubes to dry for 5 minutes.

15.   Add 50uL TE w/RNAse added.

 

Solutions

 

Solution 1

50mM Glucose

25mMN Tris-Cl pH 8.0

10mM EDTA pH 8.0

 

Solution 2

0.2g NaOH ~ 1 nugget

2.5mL 10% SDS

22.5mL ddH₂0

 

Solution 3

60.0mL 5M Potassium Acetate

11.5mL Glacial Acetic Acid

28.5mL ddH₂0