Plasmid MaxiPrep Silica Oxide

Preparation of Silica Oxide

Method 1

Dissolve in 250 ml of water, at 100 mg/ml for 30 min. Add 150 ul of 37% HCl and autoclave for 1 hour. Add more dd water and Let silica settle overnight Remove fines by suction and reconstitute the original volume using PB.

Method 2

Silica Oxide (sigma #S5631) particles: Weigh a 50-ml Falcon tube. Mix 5 g silicon dioxide (Sigma, S5631) with 50 ml sterile water in the Falcon tube and settle the tube upright overnight. Spin at 5000 rpm for 5 min and discard the supernatant. Add ddH2O to resuspend the pellet, add NaOH to final conc of 5%. Soak for 1 hour. Spin at 5000 rpm for 5 min and remove supernatnat. Weigh the Falcon tube again and calculate the net weight of the remaining silicon dioxide. Resuspend the silica pellet in 4M NaCl or Binding Solution 1 to make a final concentration of approximately 100 mg/ml. One milligram of silicon dioxide is able to bind 3–4.5-μg DNA (3). The slurry can be stored at room temperature and be stable for over 12 months.

1g silica will bind 3~4.5 mg DNA.

Method 3

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

or Celite 545

2, Let suspensionto settle o/n, 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 1 L 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 14ºC for 10 min.
8. Filter supernatant using a coffee filter paper.

9. Add 0.5 g dry weight equivalent of SiO2 (5 ml of 100 mg/ml)
10. Add 0.6 volume isopropanol, 3.3% CTAB to final conc 0.1%. Mix. Leave it at room-temperature for 15 min to 1 hour.
11. Spin at 5000 rpm at 25 ºC for 10 min.
12. Resuspend pellet in 10 ml PB, transfer to 50 ml clean centrifuge tube

13. Spin 1 min at 5000 rpm.

14. Wash again with 5 ml PB.

15. Wash 2X with 5 to 10 ml PE, resuspend everytime time

16. Add 1.5 ml preheated to 80 oC TE to suspend. Incunate in 80 oC water for 5 min

17. Spin and collect eluted DNA, transfer liquid containing DNA to a 1.5 ml tube for storage.

18. Add another hot 1.5 ml to elute again. Check OD260 to see if DNA is completely eluted.
19. Measure DNA concentration and run 5 ul on agarose gel to check for the presence of RNA.

The problem of this method is: too difficult to get rid of RNA

P3 (~5.5) [500 mL]
147g KOAc (3M)
55 mL glacial HOAc
PE: 20 mM Tris-Cl pH 7.5, 2 mM EDTA, 0.4 M NaCl, and 50% v/v ethanol

Removal of RNA

CaCl₂ Precipitation

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.

SiO2_Plasmid_binding.JPG

1	2	3	4	5	6	7
Without Ca ppt isopropanol ppt	supenatant of 0.5 M CaCl2, ppt with isopropanol	pellet of 0.5 M CaCl2 precipitation	supenatant of 1.0 M CaCl2, ppt with isopropanol	pellet of 1.0 M CaCl2 precipitation	supenatant of 1.5 M CaCl2, ppt with isopropanol	pellet of 1.5 M CaCl2 precipitation

Acid Treatment of SiO2

Add  0.1N HCl to silica oxide suspension, autoclave 30 min. Wash with water, use 50 ul to bind plasmid DNA from 300 ul of cleared lysate.

7/7/2014

use acid washed SiO2

1: +0.6 vol of isopropanol to bind to HCl washed SiO2

2: + 0.6 vol of isopropanol + 0.4% CTAB to bind to HCl washed SiO2

3:  precipitate with isopropanol

7/8/14

1	No isopropanol + 0.1%CTAB, bind directly to SiO2
2	15% isopropanol + 0.1%CTAB, bind to SiO2
3	30% isopropanol + 0.1%CTAB, bind to SiO2
4	45% isopropanol + 0.1%CTAB, bind to SiO2
5	60% isopropanol + 0.1%CTAB, bind to SiO2
6	60% isopropanol + 0.1%CTAB + 50 mM NaH2PO4, bind to SiO2
7	60% isopropanol + 50 mM NaH2PO4, bind to SiO2
8	60% isopropanol, no SiO2

Disslove Isopropano precipitated DNA with PB, mix with 50 ul acid washed SiO2.

[[Image:SiO2 plasmid 7-8 14 2.JPG

2	use spin column to wash and elute
1	use tube (no spin column)
3	control, DNA before mixing with SiO2

Large scale: from 250 ml culture

1	large scale
2	mini scale

(NH₄)₂SO₄ Salt-Out

This is somebody's work, but I can not get it to work. After (NH4)2SO4 ppt, the supernatnat can not be processed with isopropano. Adding isopropanol will for large precipitate containing salt. Salt from 300 ul cleared lysate can be dissolved by 200 ul H2O

Lane	2	3	4	5	6	7	8	9	10	11	12
(NH4)2SO4 Conc	0	0.4	0.8	1.2	1.6	2.0	2.4	2.8	3.2	3.6	4

CTBA precipitate

Cetyltrimethylammonium bromide (CTAB) is a nonionic detergent that can precipitate nucleic acids and acidic polysaccharides from low ionic strength solutions (CTAB will precipitate very little plasmid DNA from P1+P2+P3 solution). Meanwhile, proteins and neutral polysaccharides remain in solution under these conditions. In solutions of high ionic strength, CTAB will not precipitate nucleic acids and forms complexes with proteins. CTAB is therefore useful for purification of nucleic acid from organisms which produce large quantities of polysaccharides such as plants and certain Gram-negative bacteria, also remove endotoxin.

7/6/2014

Lane	Sample
1, 15	0.4% CTAB precipitate
2, 16	isopropanol ppt
3,17	60% isopropanol ppt
4	0.25M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, ppt
5	0.25M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, supernatant, add isopropanol to ppt DNA
6	0.5M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, ppt
7	0.5M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, supernatant, add isopropanol to ppt DNA
8	0.75M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, ppt
9	0.75M CaCl2, 4 oC, 30 min, spin 10 min at 25 oC, supernatant, add isopropanol to ppt DNA
10	1M (NH4)2SO4, 30 min, spin 10 min at 25 oC, ppt
11	1.5M (NH4)2SO4, 30 min, spin 10 min at 25 oC, ppt
12	2M (NH4)2SO4, 30 min, spin 10 min at 25 oC, ppt
13	1.5M (NH4)2SO4, 30 min, spin 10 min at 25 oC, supernatant, ppt with 0.4% CTAB
14	2M (NH4)2SO4, 30 min, spin 10 min at 25 oC,  supernatant, ppt with 0.4% CTAB

experiment 2

Lane	Sample
1	Isopropanol precipitate
2	0.5 M CaCl2 ppt
3	0.5 M CaCl2+ 60% isopropanol ppt
4	0.5M CaCl2 ppt
5	(0.4% CTAB + 0.5 M CaCl2) ppt
6	Supernatant of #5 (0.4% CTAB + 0.5 M CaCl2), ppt with isopropanol
7	0.4% CTAB ppt
8	supernatant of #7, ppt with isopropanol
9	0.8% CTAB ppt
10	supernatant of #9, ppt with isopropanol

the disapearence of DNA from CTAB samples is probably due to washing with 70% ethanol after CTAB precipitation. When CTAB is present DNA could be soluble in 70% EtOH

Miniprep

P1,  P3, PB are the same as Qiagen kit.

MP2: Modified P2: 0.2N NaOH, 4% Triton X-100.

1.Harvest 5 mL of overnight cultures of Escherichia coli clones of interest in Eppendorf tubes by centrifugation at 3000 rpm for 5 min and completely remove the supernatant.

2. Resuspend the pellet in 100 ul of P1 by vortexing.

3. Add 100 ul of MP2 to lyse cells. Incubate for 3~5 min at room temperature.

4. Add 100 ul of P3,  mix to form white particles. Put tube into -20°C metal block for 1~2 min.
5. Centrifuge at 13000 rpm 2 X 2.5 min. Carefully transfer supernatant to a new tube.

6. Add 500 ul of 6 M NaI.

7. Add 20 ~ 50 ul of silica particle slurry. Incubate at RT for 5~10 min (up to 20 min).

8. Spin at 13000 rpm for 1 min, discard the supernatant.

8. Wash once with 300 ul PB.

9. Wash twice with 350 ul of Washing Buffer (or 3 times with 200 ul WB). (you can also transfer silica slurry to an old Hangzhou plasmid spin column for washing).

WB: 50% EtOH. 100 mM NaCl, 10 mM Tris-HCl, pH 7, 1 mM EDTA.

10. Spin at 13000 rpm for 1 min and remove liquid.

11. Add 50 or 100 ul water, TE or EB, soak for 2 min at 70 °C.

12. Spin at 13000 rpm for 1 min, transfer liquid containing DNA to a new tube.

13. Measure DNA concentration using nanodrop.

Ref: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2829548/

Protocol 1

Silica Oxide(sigma): Dissolve in 250 ml of water, at 50 mg/ml for 30 min. Remove fines by suction and reconstitute the original volume. Add 150 ul of 37% HCl and autoclave.

Silica particles: Weigh a 50-ml Falcon tube. Mix 5 g silicon dioxide (Sigma, S5631) with 50 ml sterile water in the Falcon tube and settle the tube upright overnight (see Note 1). Remove the upper fraction containing fine silica particles and resuspend the pellet in 50 ml sterile water. Resettle the tube upright overnight. After discarding the supernatant, weigh the Falcon tube again and calculate the net weight of the remaining silicon dioxide. Resuspend the silica pellet in sterile water to make a final concentration of approximately 100 mg/ml. One milligram of silicon dioxide is able to bind 3–4.5-μg DNA (3). The slurry can be stored at room temperature and be stable for over 12 months.

P1 and P2 are the same as other kits.

P3: 3 M KoAc, pH 5.5. Chill to 4 oC before use

- 250 ml: 73.61 g Potassium acetate, dissolve in 200 ml dH2O and adjust the pH to 5.5 by adding HCl (37%), add dH2O until a total volume of 250 ml

P4: 2.5% SDS in isopropanol (SDS will not dissolve).

Acetone or 75% isopropanol

2X DNA binding solution: 6 M NaI (Sigma, 217638-500G). Filter the solution to remove impurities and store in a dark container at 4°C (see Note 2).
Washing solution: 80% (v/v) ethanol, 10 mM Tris–HCl, pH 7.5,100 mM NaCl, 1 mM EDTA.

1.Harvest 1.5-5 mL of overnight cultures of Escherichia coli clones of interest in Eppendorf tubes by centrifugation at 1000g for 5 min and completely remove the supernatant (see Notes 2-4).

2. Resuspend the pellet in 200 ul of P1 by vortexing (see Note 5).

3. Add 200 ul of P2 and invert the tube four to eight times to mix the contents (see Note 6). Incubate for 5-6 min at room temperature (see Note 7).

4. Add 200 ul of ice-cold P3 and invert the tube four to eight times (see Note 6). Incubate at -20°C for 5 min.
5. Centrifuge at 20,000g (or maximum speed in a microfuge) for 10 min to pellet the cell debris. Carefully remove the supernatant containing the plasmid DNA (see Note 8).

6. Add 120 ul of P4 (see Notes 9 and 10) and mix by vortexing. Incubate at -20°C for 20 min (see Note 11).

7. Centrifuge at 20,000g at 4°C to remove the precipitate and transfer the supernatant into a new Eppendorf tube (see Note 8).

8. Add 150 ul of prepared silica oxide solution (see Note 10). Vortex the tube to resuspend the fines and incubate for at least 5 minutes to 20 min at room temperature.

9. Centrifuge at 10,000g for 5 min and discard the supernatant (see Note 12).

10. Resuspend the pellet in 400 ul of acetone or 75% isopropanol, by vortexing. Centrifuge at 10,000g and remove the supernatant.

11. Repeat the acetone (or 75% isopropanol) wash step.

12. Dry the pellet using a Speed-vac and then resuspend in 70-90 ul of sterile H2O by vortexing for approx 1 min (see Notes 13 and 14).

13. Centrifuge at 20,000g for 5 min and recover the supernatant containing high-purity plasmid DNA.

	Culture	P1	P2	P3	P4	Silica Oxide, ml	Acetone	H2O, ul
Midi	10-25 ml	1.5	1.5	1.5	0.9	1.2	4	150-200
Maxi	50-100 ml	6	6	6	3.6	4.8	15	500-750
Mega	250-500 ml	70	70	70	42	45	100	5~6 ml

100 mL bacteria culture:  240 mg silica oxide

500 mL bacteria culture: 2.25 g silica oxide

Note:

1. To facilitate pipetting, acetone should be stored at -20°C.

2. This method can be scaled up for larger cultures; recommended volumes of solutions to use in the different formats are given in Table 1. For maxipreps or megapreps, it is advis­able to extend step 6 to 30-45 min. A version of this protocol for a 96-well format is described in ref. 5.

3. All centrifugation steps should be performed in a refrigerated centrifuge in order to pro­duce more compact pellets.

4. The bacterial pellets can be stored at -20°C after complete removal of the growth media.

5. Complete resuspension of the pellet in P1 is essential for complete subsequent lysis of the bacteria.

6. Do not vortex the lysate at this stage.

7. Do not extend the lysis time, as genomic DNA will be released from the bacteria.

8. At this stage, the supernatant can be stored at -20°C.

9. For addition of P4, avoid clogging of pipet tips by cutting off their ends to produce a wider opening.

10. Mix P4 and silica oxide stock solutions before use, as SDS and silica oxide sink to the bottom of the solution during storage.

11. Whether the DNA solution freezes or stays liquid during the -20°C incubation has no effect on the yield of plasmid DNA.

12. Do not increase the centrifugation speed of the silica oxide beyond 10,000g, as this may damage the DNA.

13. It is important to dry the silica pellet thoroughly to obtain a good yield of plasmid DNA. Once dry, the plasmid DNA can be stored at -20°C.

14. Elution of the DNA from the silica is facilitated by incubation of the resuspended silica pellet in water pre-warmed to 50°C.

Protocol 2

P1: 50 mM Tris-HCl, 10 mM EDTA, pH 8.0, 50 ug/ml RNaseA (Sigma R4642), Store at 4 C
P2: 0.2M NaOH, 1% SDS (4 g NaOH, 5 g SDS and distilled water to 500 ml)
N3: 4.2 M guanidine hydrochloride and 0.9 M potassium acetate, pH 4.2

or 1 M Guanidine Hydrochloride
WS: 100 mM NaCl, 10 mM Tris-HCl, 80% ethanol, pH 7.5
EB: 10mM Tris-HCl pH 8.5, autoclave and remeasure the pH

1. Harvest 5 ml bacterial cells by centrifuging 1-2 min, 10,000-13,000 rpm.
2. Discard supernatant (aspirate or pour off) and resuspend pellet in 250 uL P1 by vortexing.
3. Add 250uL P2 and invert 5 times to mix (wait at least 2 min but no longer than 5 min). Do NOT vortex.
4. Add 350uL N3 and invert 5 times to mix.

If P1 without RNAse is used, add 10 ul of an RNaseA solution (1.7 mg/mL) and incubate for 5 min at
room temperature.
5. Put on cold metal block (-20oC) for 5 min, or dry ice for 1 min.

6. Spin 13,000 rpm for 5 min to pellet. Transfer the supernatant to a fresh tube.

If endotoxin should be removed (or reduced):

Add 100 uL buffer F4 (2.5% SDS in isopropanol) to precipitate LPS. Mix by votex, incubate at 4°C for 5 min then -20°C for 15 min. Spin for 10 min at maxispeed (microcentrifuge)

7. Add 25 µl silica matrix, mix well and let the tube sit at room temperature for 2 min.
8. Pellet the matrix by centrifugation for 20 sec at 13,000 rpm. Pour off the supernatant and gently tap the
inverted tube against a paper towel to drain the liquid.
9. Wash the matrix by resuspending with 750 µl WS and vigorously vortexing.
10. Repeat step 7 and 8 to wash glass matrix again.
11. Pellet the matrix by centrifugation for for 20 sec at 13,000 rpm. and remove the supernatant by pipetting.
12. Slowly blow air to dry the matrix (it takes less than 1 min), or you can leave the tube on bench for 10-15
minutes.
13. Add 55 µl EB to resuspend the pellet by brief vortexing and place the microfuge tube at 80°C for 2 min.
14. Centrifuge at 13,000 rpm for 2 min and transfer ~50 µl supernatant containing the eluted plasmid DNA to another tube. Apply DNA to downstream reaction or store at -20°C.

Hangzhou Column

P1: 50 mM Tris-HCl, 10 mM EDTA, pH 8.0, 50 ug/ml RNaseA (Sigma R4642), Store at 4 C
P2: 0.2M NaOH, 1% SDS (4 g NaOH, 5 g SDS and distilled water to 500 ml)
N3: 4.2 M guanidine hydrochloride and 0.9 M potassium acetate, pH 4.2

PB:  5 M guanidine hydrochloride, 30% isopropanol or ethanol

ETR: 20% Triton X-114 in ethanol or isopropanol
PE: 100 mM NaCl, 10 mM Tris-HCl, 80% ethanol, pH 7.5
EB: 10mM Tris-HCl, pH 8.5, autoclave and remeasure the pH

Miniprep

Add 25 ul of silica slurry to Hangzhou spin column

1. Harvest 5 ml bacterial cells by centrifuging 1-2 min, 10,000-13,000 rpm.
2. Discard supernatant (aspirate or pour off) and resuspend pellet in 250 uL P1 by vortexing.
3. Add 250uL P2 and invert 5 times to mix (wait at least 2 min but no longer than 5 min). Do NOT vortex.
4. Add 350uL N3 and invert at least 5 times to mix.
5. Spin 13,000 rpm for 5 min to pellet. At the same time, please prepare EB to be heated at 60°C.

6. Equilibrate a spin column  with 100 ul 10% NaOH for 5 min, spin to remove residual NaOH.
7. Transfer the supernatant onto a preequilibrated spin column, centrifuge at 8,000rpm for 1 min, discard the flow-through; or use the vacuum manifold.
8. Add 750 ul PE buffer to wash the column, centrifuge at 8,000rpm for 1 min; or use the vacuum manifold.
9. Repeat step 8.
10. Centrifuge again for 2 minutes to remove the residual ethanol, discard the flow-through. Or use the vacuum manifold to remove the residual ethanol.
11. Set the column on a new 1.5mL microcentrifuge tube.
12. To elute DNA, add 50 ul warm EB Buffer onto the membrane, and place the column and microfuge tube at 80°C for 1 minute.
13. Centrifuge at 13,000 rpm for 1 min to elute the DNA. Apply DNA to downstream reaction or store at -20°C.

Endofree Maxiprep

1. Grow 250 ml to 1 L of bacterial culture to an OD600 of 2-3.
2. Harvest bacteria cells by centrifugation for 10 min at 6000 rpm. Discard the supernatant.
3. Resuspend pelleted cells in 15 ml of P1 with RNase A. The bacterial pellet can be resuspended by vortexing, use a clean plastic spoon to disrupt,  or pipetting up and down until no cell clumps remain.
4. Add 15 ml of P2 and mix gently by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. If it is very sticky, add more P1 and P2 proportionally,  Incubate for 3 min at room temperature.
Note. Do not vortex to avoid shearing chromosomal DNA. Do not incubate for more than 3 min to avoid denaturation of supercoiled plasmid DNA.
5. Add 15 ml of N3 and mix immediately by inverting the tube 5-8 times.

Cap the tube and gently mix by inverting tube several times until flocculent white precipitates form. This may require a 2 minute incubation at room temperature with occasional mixing.

Note: The solution must be mixed thoroughly. This is vital to obtaining good yields. If the mixture still appears viscous, brownish or conglobated, more mixing is required to completely neutralize the solution.

6. Centrifuge for 10 min at 12,000 rpm to pellet cell debris and chromosomal DNA. Filter supernatant using a coffee filter.

Note: You can add 2 ~ 5 ml P1 with RNase and incubate o/n at RT, then centrifuge,  RNA will be degraded completely.

2012/12/7: add 2 ml of P1 with RNase, incubate o/n at RT, RNAse is degraded:

7. Preequilibrate column with 3 ml of 10% NaOH for 5 min at RT. Spin to remove residual NaOH.

8. Add 1/10 vol of 20% Triton X-114 in ethanol to filtered DNA solution.

9. Load DNA solution onto the spin column.

10. Spin at 3000 rpm for 1 min.

12. Wash column with 10 ml PB Buffer. Spin for 1 min at 3000 rpm

13. Wash column 2 times with 10 ml PE. Spin for 1 min at 3000 rpm

14. Spin at 3000 rpm for 3 min.

15. Elute DNA 2 times with 0.5 ml TE or EB.

16. Measure DNA concentration and OD260/280 ratio.

17. QC DNA by digestion and transfection

11/29/2012

Sample	#528	#528	#528	#528	#528	#529	#529	#529	#529	#529	#529
vol	0.5 ml	0.5 ml	0.5 ml	miniprep	miniprep	0.5 ml	0.5 ml	0.5 ml	0.5 ml	0.5 ml	0.5 ml
elute	1st	2nd	3rd			1st	2nd	1st	2nd	3rd	4th
OD260	224	329	115	204	104	83	86	321	196	122	93
260/280	1.84

Protocol 3

Briefly, 5 g silicon dioxide (Sigma, S5631) was mixed with 50 ml sterile water in a 50 ml Falcon tube and settled for 2 h. The supernatant, containing fine silica particles with sizes below ca. 1 μm, was removed and the pellet was resuspended in 50 ml sterile water and re-settled for another 2 h. After discarding the supernatant, the packed silica was resuspended in 50 ml sterile water to make a final concentration of approximately 100 mg/ml. One mg of the silicon dioxide is able to bind 3-4.5 μg DNA. The slurry can be stored at room temperature and will be stable for at least 12 months.

Solution A: (Resuspension solution) 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 100 g/ml RNase A

Solution B: (Alkaline lysis solution) 0.2 M NaOH, 1% sodium dodecyl sulfate (SDS)

Solution C: (Neutralization solution) 1.32 M KOAc, pH 4.8

Solution D: (NaI solution) 6 M NaI, or 5 M NaCl

Solution E: (Washing solution) 50% ethanol, 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA

Solution F: 10% SDS

Solution G:  Phenol:chloroform:isoamyl alcohol (25:24:1, v/v)

1. Pellet the cells of 2 ml E. coli overnight culture (OD600 = 2.0) growing at 37°C or A. tumefaciens overnight culture (OD600 = 2.0) growing at 28°C by centrifugation at 16,000 g for 30 sec.

2. Resuspend the cell pellet in 100 μl Resuspension solution (Solution A) by brief vortexing.

3. Add 100 μl Alkaline lysis solution (Solution B) and invert the microfuge tube for several times.

4. Let the tube sit for 2 min for E. coli or 5 min for A. tumefaciens at room temperature.

5. Add 100 μl Neutralization solution (Solution C) and invert the tube for a few times.

6. Centrifuge at 16,000 g for 5 min and transfer the supernatant to a fresh tube containing 500 μl 6 M NaI solution (Solution D) and mix well by inverting the tube.

7. Add 20 μl silica matrix, mix well and let the tube sit at room temperature for 2 min.

NOTE: Extending the incubation time to 5 min could slightly increase the yield.

8. Pellet the matrix by centrifugation for 10 sec at 16,000 g. Pour off the supernatant and gently tap the inverted tube against a Kimwipe to drain the liquid.

9. Wash the matrix by resuspending with 500 μl Washing solution (Solution E) and vigorously vortexing.

10. Repeat step 8 and 9.

11. Pellet the matrix by centrifugation for 10 sec at 16,000 g and remove the supernatant by pipetting.

12. Centrifuge for another 10 sec and carefully pipette off the residual liquid.

13. Add 40 μl sterile water to resuspend the pellet by brief vortexing and place the microfuge tube at 70°C for 2 min.

14. Centrifuge at 16,000 g for 2 min and transfer 36-38 μl supernatant containing the eluted plasmid DNA to a fresh tube.

NOTE: The remaining 2-4 μl liquid should be abandoned due to a slight contamination by disturbed silica particles. For protection against DNase digestion or pH fluctuations, TE buffer (10 mM Tris-Cl, pH8.0, 1 mM EDTA) should be used to elute DNA.

Sub-protocol 2 (For DNA purification from solution or agarose gel slice)
1. Add 150 μl 6 M NaI (Solution D) to up to 50 μl PCR or restriction digestion mixture and mix well by inverting the tube. In case of the agarose gel slice containing DNA fragments, add 300 μl Solution D for every 100 mg gel slice and heat the microfuge tube at 70°C for 3 min to dissolve the gel.

2. Add 10 μl silica matrix to the blend, mix well and incubate for 2 min at room temperature.

NOTE: Extending the incubation time to 5 min could slightly increase DNA recovery rate.

3. Pellet the matrix by centrifugation for 10 sec at 16,000 g and remove the supernatant by pipetting.

4. Wash the matrix in 500 μl Washing solution (Solution E) by vigorously vortexing.

5. Repeat step 3 and 4.

6. Pellet the matrix by centrifugation for 10 sec at 16,000 g and discard the supernatant.

7. Centrifuge for another 10 sec and pipette off the trace amount of liquid.

8. Resuspend the matrix in 5-30 μl sterile water and place the microfuge tube at 70°C for 2 min.

9. Centrifuge at 16,000 g for 2 min and transfer the DNA eluate into a fresh tube.

NOTE: For protection against DNase digestion or pH fluctuations, TE buffer (10 mM Tris-Cl, pH8.0, 1 mM EDTA) should be used to elute DNA.

Sub-protocol 3 (For plant genomic DNA extraction)
1. Place approximately 10 mg of plant material in a 1.5 ml microfuge tube.

2. Add 200 μl Resuspension solution (Solution A) and grind the tissue using a Micro-Grinder homogenizer (Research Products International Corporation).

3. Add 30 μl 10% SDS (Solution F) to the homogenate and invert the tube for several times.

4. Incubate the tube at 70°C for 10 min.

5. Add 250 μl Phenol:chloroform:isoamyl alcohol (25:24:1, v/v; Solution G) and vortex the mixture vigorously for 30 sec.

6. Centrifuge at 4°C at 16,000 g for 5 min and transfer the aqueous (upper) phase to a fresh tube containing 500 μl 6 M NaI (Solution D) and mix well by inverting the tube.

7. Add 20 μl silica matrix and let the tube sit at room temperature for 2 min.

8. Pellet the matrix by centrifugation at 16,000 g for 10 sec and remove the supernatant by pipetting.

9. Wash the matrix by resuspending with 1 ml Washing solution (Solution E) and vigorously vortexing.

10. Repeat step 8 and 9.

11. Pellet the matrix by centrifugation at 16,000 g for 10 sec and remove the supernatant using a pipette.

12. Centrifuge for another 10 sec and carefully pipette off the residual liquid.

13. Add 40 μl sterile water to resuspend the pellet by brief vortexing and place the microfuge tube at 70°C for 2 min.

14. Centrifuge at 16,000 g for 2 min and transfer 36-38 μl supernatant containing the eluted genomic DNA to a fresh tube.

NOTE: The remaining 2-4 μl liquid should be abandoned due to a slight contamination by disturbed silica particles. For protection against DNase digestion or pH fluctuations, TE buffer (10 mM Tris-Cl, pH8.0, 1 mM EDTA) should be used to elute DNA.