Large Scale Plasmid Preparation.

 

Solution 1. 50mM Glucose, 10mM EDTA, 25 mM Tris-Cl, pH 8.0 (Cold)

Solution 2. 0.2N NaOH, 1% SDS (fresh each time, use stock of NaOH and SDS).

Solution 3.  5M K Acetate, pH 4.8.  See Maniatis for the preparation (p.368, 1^st edition).

TE: 10mM Tris, pH 7.5, 1 mM EDTA.

10M Ammonium acetate

10mg/ml RNase A, DNAse free.

Lock Gel.,  PLG 2ml,  5prime.com

 

1.     Grow bacteria in 100 ml 2x YT media overnight. Shake at 250 rpm.

2.     Harvest cells, 5K for 10 min.

3.     Pour off all supernatant.

4.     Resuspend into 5 ml of Sol. 1 on ice.

5.     Add 20 mg lysozyme.

6.     Incubate at room temp for 5 min.

7.     Add 10 ml Sol. 2, mix by inverting 4 times. Place on ice for 5 min.

8.     Add 7.5 ml Sol. 3, mix by inverting, place on ice for 5 min.

9.     Spin 10K, 20 min.

10. Take supernatant, add 1 volume of isopropanol (2-propanol).  Mix, place on ice for 5 min.

11. Spin 10K, 20 min.

12. Wash with cold 66% ETOH, spin 10K, 5 min.

13. Remove all ethanol, let air dry.

14. Dissolve pellet in 2 x 0.4 ml TE.  Pool and add 4 ul, 10 mg/ml RNase A (DNase Free). Incubate 30 min at 37oC.

15. Divide into 2- 1.5 ml micro tubes.  Add 0.4 ml of phenol-chloroform, vortex well for 15 sec.  Set aside.

16. Get 2 Phase Lock PLG 2ml (5Prime.com), pre-spin 2 tubes for 20-30 sec from 12-16K x g.

17. Add 0.75 ml of the aqueous/phenol phase to the Phase Lock tubes.

18. Spin for 5 min from 12-16K x g.

19. Take supernatant and add 0.1 ml 10M NH₄Ac.

20. Add 1 ml ETOH and mix by inverting. Ice for 10 min.

21. Spin at high speed for 10 min.

22. Wash 1 time with 1 ml 66% ETOH.

23. Remove supernatant, air dry.  Dissolve in 0.5 ml TE.  Measure OD 260.  Take 5 ul into 1 ml TE.  If 1 cm cuvette, then the absorbance x 10 = concentration in mg/ml.  Check DNA by restriction digestion.  There should be no RNA.