Editing Plasmid MaxiPrep Hydroxyapatite (section)

== Purify Plasmid DNA from Isopropanol Precipitate<br>  ==

1. If Qiagen anion exchange column is not available, use 0.6 vol isopropanol to precipitate plasmid DNA from lysate (P1 + P2 + P3).<br> 

Alternatively, DNA will absorb to acid washed autoclaved SiO2 particles when the lysate is mixed with 15%~60% isopropanol, 0.1% CTB after incubating for 30 min. Spin down SiO2 particles and wash 2 times with PE, then elute plasmid DNA&nbsp; with 2 to 3 times of 1.5 ml hot (80 oC) TE/EB. Before loading into&nbsp;CHT Ceramic Hydroxyapatite column, the DNA solution shold be mixed with 9 vol of 7~8 M urea, 0.24 M sodium phosphate buffer pH 6.8, 0.1% SDS or CTAB<br> 

2. Add 0.6 vol of isopropanol, incubate at RT for 10 ~15 min, spin at 5000 rpm for 10~15 min, rinse pellet once with 70% EtOH to remove as much of acetate as possible.<br> 

3. Dissolve pellet in 10 ml '''Binding Buffer: '''8 M urea, 0.24 M sodium phosphate buffer pH 6.8~7.0, '''0.1% SDS or CTAB'''.<br> 

4. Mix solubilized solution with 1 g&nbsp; Bio-Rad CHT Ceramic Hydroxyapatite Type II Media (1 g powder will be enough to purify plasmid DNA from 1 L culture). Incubate at RT for 15 min with ocassional mixing.<br> 

5. Spin and decant supernatant carefully.&nbsp; Wash the particles with 10 ml of '''Washing Buffer 1''': 8 M urea, 0.24 M phosphate buffer, pH 6.8. Spin down particles, if necessary wash with&nbsp;Washing Buffer 1&nbsp; until the absorbance at 260 nm of supernatant is approximately zero.<br> 

6. Wash with ~45 ml of '''Washing Buffer 2''': 10 mM phosphate buffer, pH 6.8 (to remove urea). Spin and remove supernatant.<br> 

7. Add ~10 ml Washing Buffer 2 and transfer particles to a Pierce spin column. 

8. Spin 3000 rpm for 5 min to remove residual washing buffer. 

9. Add 1 ml&nbsp; '''Elution Buffer''': 0.3&nbsp; M phosphate buffer, pH 6.8, soak 1 min, spin at 3000 rpm for 5 min and collect 1&nbsp; ml fractions, continue to elute until OD260 ~&gt; 0.05. Total elution vol is about 10 ml for 1 g particles.<br> 

10. Combine fractions containing DNA. Measure OD260. 

11. Dilute DNA solution with equal volume of water, add 0.6 vol isopropanol to precipitate DNA onto a "Qiagen" precipitator (filter)<br> 

12. Elute DNA with TE or EB.<br> 

13. Measure DNA conc using nanodrop<br> 

14. Run gel to check purity (to see if there is any RNA or genomic DNA contaimination)<br> 

15. Regenerate CaPO4 particles with&nbsp; 1 N NaOH, then 1 M NaPO4 buffer, pH 7.0, wash with water. Store in 20% ethanol. 

'''7/9/14'''<br> 

<u>'''Binding of plasmid to&nbsp; HTP particles'''</u><br> 

[[Image:Hydroxylapatide plasmid 7 9 14 2.JPG]]<br><br> 

{| width="496" cellspacing="1" cellpadding="1" border="1"
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| <br> 
| 1<br> 
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| 7<br>
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| HTP types<br> 
| Bio-Rad HTP <br> 
| Bio-Rad HTP 
| Bio-Rad Ceramic HTP type 1&nbsp; <br> 
| Bio-Rad Ceramic HTP type 1 
| Bio-Rad HTP DNA Grade <br> 
| Bio-Rad HTP DNA Grade 
| Plasmid DNA pellet solubilized in Binding Buffer<br>
|-
| Phosphate Conc.<br> 
| 0.24M NaPO4<br> 
| 0.12M NaPO4 
| 0.24M NaPO4 
| 0.12M NaPO4 
| 0.24M NaPO4 
| 0.12M NaPO4 
| <br>
|}

Bio-Rad Ceramic HTP type 1 does not bind plasmid DNA very well<br> 

'''<u>Plasmid Eluted from HTP particles</u>'''<br> 

[[Image:Hydroxylapatide plasmid 7 9 14.JPG]]<br> 

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| Bio-Rad HTP 
| Bio-Rad Ceramic HTP type 1 
| Bio-Rad HTP DNA Grade 
| Plasmid DNA pellet solubilized in Binding Buffer<br>
|}

7/11/14<br> 

maxiprep <br> 

[[Image:CaPO4 plasmid 7 11 14.JPG]]<br> 

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| Bio-Rad HTP<br> 
| Bio-Rad HTP DNA Grade <br> 
| Bio-Rad HTP<br> 
| Bio-Rad HTP DNA Grade <br>
|-
| Clone 470<br> 
| 470<br> 
| 471<br> 
| 471<br>
|}

<br> 

<br> 

'''For bioling plasmid protocol:'''<br> 

Resuspend E coli in 10 mM Tris, 10 mM NaCI, 2 mM EDTA, pH 7.6. Heat the suspension at 100 "C for 2 min, then rapidly freezing to - 70 "C in dry-ice /EtOH bath. Warm up and spin to remove pellet. Mix supernatant with 9 vol of 7~8 M urea, 0.24 M sodium phosphate buffer pH 6.8, 0.1% SDS or CTAB.<br> 

<br> 

'''For lysome protocol:'''<br> 

For 1 L culture<br> 

Resuspend E coli in 4 ml of 0.25 M sucrose, 2 mM MgC12<br> 

Add 0.8 ml (1/5 volume) of lysozyme solution&nbsp; 20 mg/ml lysozyme (Sigma Ltd) in 10 mM Tris/HCl, pH 8.0.<br> 

Incubate for 5 min on ice<br> 

Add 1.6 ml (2/5 volume of inital volume) of&nbsp; 0.25 M EDTA, pH 8.0<br> 

Incubate for 5 min on ice <br> 

Add 6.4 ml (equal vol) of&nbsp; 0.5&nbsp;% (w/v) Triton X-100, 62.5 mM EDTA, 50 mM Tris-HCl, pH 8.0<br> 

Incubate for 20 rnin on ice, swirl every 5 min.<br> 

Spin and collect supernatant, mix cleared solution with 9 vol of 7~8 M urea, 0.24 M sodium phosphate buffer pH 6.8, 0.1% SDS or CTAB.<br> 

<br> 

=== Regeneration and Storage of Bio-Rad CHT Ceramic Hydroxyapatite Type II.<br>  ===

<br>0.5M sodium phosphate, pH 7<br>1 M trisodium phosphate, pH 11–12<br> 

<br>Autoclave: 121°C, 20 min in phosphate buffer, pH 7<br> 

Sanitization: 1–2 N NaOH<br>Recommended column storage: 0.1 M NaOH<br>