Paul.xu at 17:03, 4 July 2014

2014-07-04T17:03:11Z

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== Preparation of diatomaceous earth (Sigma D-5384) ==

1, Mix 30 g of diatomaceous earth with 300 ml of sterile milliQ water

or Celite 545 

2, Let suspensionto settle for 2 to 3 hours, discard upper liquid phase discarded.

3, Resuspend the settled particulate matter in 60.0 ml of sterile milliQ water. This yield stock solution of approximately 150 mg/ml (w/v density of 1.130 to 1.150 g per ml).

4, Add 2.0 ml suspension to 50 ml of chaotropic '''Binding Solution 1''' (7 M guanidine HCl in 50 mM Tris; 20 mM EDTA; pH 7.0, or 6 M guanidine thiocyanate in 50 mM Tris; 20 mM EDTA; pH 7.0)

'''Binding Solution 2: ''' 

6.0 M sodium perchlorate. 0.05 M Tris-Cl pH 8, and 10.0 mM ethylenediaminetetraacetic acid (EDTA). 

5. or mix 5 g of diatomaceous earth with 50 ml of chaotropic binding solution.

== MaxiPrep ==

1. Inoculate 1L mL 2xYT or LB/AMP or Kan from 1-5 mL overnight 2. Grow at 37 oc overnight. 3. Spin cells at 6000 rpm for 10 min at 4ºC using GS3 rotor. 4. Resuspend cells in 20 mL P1 without RNase A. 5. Add 20 mL P2. Stir very gently using a long 5 ml plastic pippette, until homogenous and clear. 6. Add 20 ml P3, mix, add 100 mg/ml RNAse sol to 0.1 mg/ml. Leave it on bench to digest o/n 7. Spin 6000 rpm at 4ºC for 10 min. 8. Filter supernatant using a coffee filter paper. 10. Add 4 M CaCl2 to final conc of 0.5 M, incubate at RT for 10 min. Spin at 25 oC (RT) for 10 min at 5000 rpm. 11. Add 0.6 volume isopropanol or 2 volume of ethanol. Invert to mix. Leave it at room-temperature for 10 min. 12. Spin at 6000 rpm at 4ºC or 25 ºC for 10 min. 13. Resuspend pellet in 5X1 ml PB,  combine liquid and transfer to a clean 50 ml tube. (14. Add 10% Triton X-110 to final concentration of 0.1% TritonX-114 to the plasmid DNA solution, mix by votexing.) 15. Add 5 ml stock SiO2  slurry. Incubate at RT for 1 hour. 20. Spin down SiO2 particles at 5000 rpm for 5 min. 21. Wash SiO2 particles with 2 times PB buffer than 2 times with 15 ml of '''Washing Solution (PE). Spin''' at 5000 rpm for 5 min. 

PE: 20 mM Tris-Cl pH 7.5, 2 mM EDTA, 0.4 M NaCl, and 50% v/v ethanol 

 22. Wash SiO2 particles with 5 ml of 80% ethanol. Spin at 5000 rpm for 5 min and remove liquid 23. Add 2 ml of water or TE preheated to 65-70 oC, and wait 2 min (or place on hot bath for 2 min) 24. Spin at 5000 rpm for 5 min. 25. Transfer liquid containing DNA to a 1.5 ml tube for storage. 26. Measure DNA concentration and run 5 ul on agarose gel to check for the presence of RNA.

 '''P3 (~5.5) [500 mL]''' 147g KOAc (3M) 55 mL glacial HOAc 

Precipitate RNA using CaCl2. Add 43 ul, 100 ul and 120 ul of 4 M CaCl2 to 300 ul filter solution from step 8, incubate at RT for 10 min, spin using microcentrifuge for 10 min. 

Transfer supernatant to another tube. Add 300 ul isopropanol. incubate at RT for 15 min on bench, spin down pellet. Dissolve both Ca ppt and isopropanol ppt in 50 ul TE. Use 8 ul to run gel.

[[Image:Ca_ppt_RNA-7_4-14.JPG]] 

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Without Ca ppt

isopropanol ppt 

| supenatant of 0.5 M CaCl2, ppt with isopropanol 
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| pellet of 1.0 M CaCl2 precipitation 
| supenatant of 1.5 M CaCl2, ppt with isopropanol 
| pellet of 1.5 M CaCl2 precipitation 
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== Protocol 2 ==

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

 

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&nbsp;

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

4. Add 30 ml P3 (3 M sodium acetate pH 5.2). mix&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 &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&nbsp;EDTA, 0.05% Triton X-100)

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

13. Transfer to spin column.

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

15. Spin&nbsp;10 min to dry out

16. Add 5 ml&nbsp;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 MaxPrep DNA using Diatomaceous Earth - Revision history

Paul.xu at 17:03, 4 July 2014