Editing Plasmid MaxiPrep Silica Oxide (section)

== 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''':&nbsp;(Alkaline lysis solution)&nbsp;0.2 M NaOH, 1% sodium dodecyl&nbsp;sulfate (SDS) 

'''Solution C''':&nbsp;(Neutralization solution)&nbsp;1.32 M KOAc, pH 4.8 

'''Solution D''':&nbsp;(NaI solution)&nbsp;6 M NaI, or 5 M NaCl 

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

'''Solution F''': 10% SDS 

'''Solution G''': &nbsp;Phenol:chloroform:isoamyl alcohol&nbsp;(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)<br>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)<br>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. 

[[Category:Protocols]]