Editing Plasmid MaxiPrep Silica Oxide (section)

== 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 ([http://www.sigmaaldrich.com/catalog/product/sial/s5631?lang=en&region=US 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. 

<br> 

P1 and P2 are the same as other kits. 

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

- 250 ml:&nbsp;73.61 g Potassium acetate, dissolve in 200 ml dH2O and adjust the pH to 5.5 by adding HCl (37%), add&nbsp;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).<br>'''Washing solution''': 80% (v/v) ethanol, 10 mM Tris–HCl, pH 7.5,'''100''' mM NaCl, 1 mM EDTA.<br><br> 

<br> 

<br> 

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&nbsp;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&nbsp;of ice-cold P3 and invert the tube four to eight times (see Note 6). Incubate at -20°C for 5 min. <br>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&nbsp;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&nbsp;of prepared silica oxide solution (see Note 10). Vortex the tube to resuspend the fines and incubate for at least 5 minutes to&nbsp;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. 

<br> 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| <br> 
| 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: &nbsp;240 mg silica oxide 

500 mL bacteria culture: 2.25 g silica oxide<br> 

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. <br> 

<br>