Editing Plasmid MaxPrep DNA using Diatomaceous Earth (section)

== Protocol 2<br>  ==

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 ddH2O 

add 2 ml of the resuspended diatomaceous earth to 50 ml 6M GnHCl 

<br> 

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&amp;nbsp; 

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

4. Add 30 ml P3 (3 M sodium acetate pH 5.2). mix&amp;nbsp; 

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 &amp;nbsp;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&amp;nbsp;EDTA, 0.05% Triton X-100) 

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

13. Transfer to spin column. 

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

15. Spin&amp;nbsp;10 min to dry out 

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

http://www.my-whiteboard.com/diatomaceous-earth-plasmid-prep/&lt;br&gt;&lt;br&gt; 

<br> 

<br> 

[[Category:Protocols]]