Paul.xu at 03:18, 1 July 2014

2014-07-01T03:18:07Z

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== Materials:  ==

Diatomaceous earth (Sigma D5509, 1 kg): D5509-1KG, $76.10

Diatomaceous earth (Sigma D5384): ~97.5% SiO2 basis, powder, discontinued

shake up 5 mg of diatomaceous earth (Sigma D5384) in 100 ml ddH2O and let it settle for 3 hours  remove the supernatant containing fine particles repeat this step add ddH2O to 100 ml (and EDTA to 20 mM if you want) and store the suspension at 4°C prepare 6 M guanidine hydrochloride (GnHCl) in ABD add 2 ml of the resuspended diatomaceous earth to 50 ml 6M GnHCl

== ==

== Protocol ==

Make semipure plasmid using standard alkaline lysis and isopropano precipitation method

1. Spin down cells from 1 L culture.

2. Resuspend in 30 ml P1 without lysozyme, use spetuler 

3. Add 30 ml P2, mix gently by inversion 2–3 times, and incubated at room temperature for 5 min to lyse cells.

4. Add 30 ml P3 (3 M sodium acetate pH 5.2). mix 

5. Add RNase, incubate at RT for 3 hours to o/n

6. Spin down pellet. Collect supernatant.

7. Add 1 ml 20% Triton X-100 in ethanol, incubate for 10 min to 1 hour at RT

8. Add 55 ml isopropanol. Incubate on ice for 30 min.

9. Spin down DNA.

10. Wash DNA with 70% ethanol.

11. Dissolve DNA in 1 to 5 ml  Milli-Q water, add 5 volume of DNA Binding Solution (DBS):

7 M guanidine hydrochloride (GuHCl) in 50 mM Tris-HCl buffer pH 6.0~7.0, and 20 mM EDTA, 0.05% Triton X-100)

12. Add 20 g Diatomaceous earth, mix and rock at a slow speed at room temperature for 45–60 min.

13. Transfer to spin column.

14. Wash twice with PE buffer (20 mM Tris-Cl pH 7.5, 200 mM NaCl, 5 mM EDTA, and 50% ethanol), then 10 ml 80% isopropanol

15. Spin 10 min to dry out

16. Add 5 ml 72 °C Milli-Q H2O, immediately spin at 3000 rpm for 5 min.

http://www.my-whiteboard.com/diatomaceous-earth-plasmid-prep/ 

[[Category:Protocols]]

Plasmid MaxiPrep using Diatomaceous Earth - Revision history

Paul.xu at 03:18, 1 July 2014