Editing Plasmid MaxiPrep Silica Oxide (section)

== Hangzhou Column<br>  ==

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

'''PB''':&nbsp; 5 M guanidine hydrochloride, 30% isopropanol or ethanol <br> 

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

'''Miniprep'''<br> 

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

6. Equilibrate a spin column&nbsp; with 100 ul 10% NaOH for 5 min, spin to remove residual NaOH.<br>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.<br>8. Add 750 ul PE buffer to wash the column, centrifuge at 8,000rpm for 1 min; or use the vacuum manifold.<br>9. Repeat step 8.<br>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.<br>11. Set the column on a new 1.5mL microcentrifuge tube. <br>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.<br>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'''<br> 

1. Grow 250 ml to 1 L of bacterial culture to an OD600 of 2-3.<br>2. Harvest bacteria cells by centrifugation for 10 min at 6000 rpm. Discard the supernatant.<br>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,&nbsp; or pipetting up and down until no cell clumps remain.<br>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,&nbsp; Incubate for 3 min at room temperature.<br>Note. Do not vortex to avoid shearing chromosomal DNA. Do not incubate for more than 3 min to avoid denaturation of supercoiled plasmid DNA.<br>5. Add 15 ml of N3 and mix immediately by inverting the tube 5-8 times.<br> 

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. 

<br>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.<br><br> 

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,&nbsp; RNA will be degraded completely. 

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

[[Image:Plasmid maxi silica dioxide 2012 12 7.jpg]] 

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. <br> 

9. Load DNA solution onto the spin column. 

10. Spin at 3000 rpm for 1 min.<br> 

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<br> 

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<br> 

<br>11/29/2012<br> 

[[Image:Silica dioxide maxiprep 2012 11 29.jpg]]<br> 

{| width="200" cellspacing="1" cellpadding="1" border="1"
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| Sample 
| #528<br> 
| #528<br> 
| #528<br> 
| #528<br> 
| #528<br> 
| #529<br> 
| #529<br> 
| #529<br> 
| #529<br> 
| #529<br> 
| #529<br>
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| vol<br> 
| 0.5 ml<br> 
| 0.5 ml 
| 0.5 ml<br> 
| miniprep<br> 
| miniprep<br> 
| 0.5 ml<br> 
| 0.5 ml<br> 
| 0.5 ml<br> 
| 0.5 ml<br> 
| 0.5 ml<br> 
| 0.5 ml<br>
|-
| elute 
| 1st 
| 2nd 
| 3rd 
| <br> 
| <br> 
| 1st 
| 2nd 
| 1st 
| 2nd 
| 3rd 
| 4th
|-
| OD260<br> 
| 224<br> 
| 329<br> 
| 115<br> 
| 204<br> 
| 104<br> 
| 83<br> 
| 86<br> 
| 321<br> 
| 196<br> 
| 122<br> 
| 93<br>
|-
| 260/280<br> 
| 1.84<br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|}

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