Editing Plasmid MaxiPrep Hydroxyapatite

== Qiagen or MN Protocol<br>  ==

Use Qiagen tip 2500 (Mega) or MN AX 2000 (Mega), or AX 10000 (Giga) columns.<br> 

Follow their protocol to elute plasmid DNA from column.<br> 

For Qiagen column add 0.1% Triton X-114 to cleared lysate and incubate on ice for 20 min before loading to column. 

For Qiagen column elute plasmid with QF buffer.<br> 

'''QF buffer '''(elution buffer) 

1.25M NaCl<br>50 mM Tris-HCl pH 8.5<br>15% isopropanol 

to make 2 L<br>833 ml 3M NaCl<br>100 ml 1M Tris pH 8.5<br>300 ml EtOH<br>dH2O to 2 liters<br>check the pH and adjust to 8.5 (it should be close) with NaOH or HCl<br>store QC and QF in tightly capped containers at room temp.<br> 

== Optional: Endotoxin and RNA reduction Protocol<br>  ==

1. Spin down cells from 1 L LB culture for 5 min at 6000 rpm<br>2. Resuspend pellet in 50 ml of cold 20% sucrose , 50 mM Tris-HCl, pH 8.0 .<br>3. Add 10 ml of freshly prepared lysozyme (5 mg/ml in 0.25 M Tris, pH 8.0)<br>4. Add 20 ml of cold 0.25M EDTA, pH 8.<br>5. Leave on ice for 5 min.<br>6. Add 20 ml of cold 0.25M Tris-HCl, pH 8.0, incubate at 37 oC for&nbsp; 30 min.<br>7. Spin at 4000 g for 10 min, remove viscous supernatant carefully.<br>8. Resuspend spheroplast pellet in 120 ml of 50 mM glucose/25 mM Tris-HCl, pH 8.0/10 mM EDTA, pH 8.0 containing 100 ug/ml RNase A, <br> 

or P1 containing purified RNAse (DNase-free RNase) or heat-treated RNase.<br> 

or 120 ml S1-EF <br> 

<br>9. Incubate at 37 oC for 30 min or room temperature o/n.<br> 

<br>10. Add 120 ml of P2 (or S2-EF) to lyse the cells.<br>11. Add 120&nbsp; ml of P3 (or S3-EF).<br>12. Spin 6000 X RPM 10 min<br>13. Collect cleared lysate, filter through a coffer paper.<br><br> 

== Plasmid Capture using MN NucleoBond AX 10000 Giga column.  ==

1. Regenerate one&nbsp; MACHEREY-NAGEL NucleoBond AX 10000 (Giga) column using 150 ml 2 M NaCl.<br> 

if flow rate is very slow, wash with 50 ml 0.1N NaOH then 200 ml water before regenerating using 150&nbsp; ml of 2 M NaCl 

2. Equilibrate column with 100 mL Buffer N2.<br> 

3. Load cleared lysate onto the column<br> 

4. Wash with 4 x 150 ml of N3-EF.<br> 

5. Wash with 3 X 130 ml of&nbsp; N4-EF.<br> 

6. Elute with 100 mL of N5-EF.<br> 

<br> 

== 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"
|-
| <br> 
| 1<br> 
| 2<br> 
| 3<br> 
| 4<br> 
| 5<br> 
| 6<br> 
| 7<br>
|-
| 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> 

{| width="436" cellspacing="1" cellpadding="1" border="1"
|-
| 1<br> 
| 2<br> 
| 3<br> 
| 4<br>
|-
| 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> 

{| width="492" cellspacing="1" cellpadding="1" border="1"
|-
| 1 
| 2 
| 3 
| 4
|-
| 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>

== Plasmid Purification using hydroxyapatite <br>  ==

Use 1 g hydroxyapatite for 1 litre of bacterial culture. This will give 3~5 ml wet beads. 

1. Weigh 2X 0.5 g hydroxyapatite powder (Biorad, DNA grade, will bind 500 to 800 ug DNA,&nbsp;Cat #130-0520, or 130-0420, non-DNA grade), add to two 50 ml tubes. Add 30 ml water.<br>Let the beads settle for 10 min. Decant to remove fine particles. 

2. Resuspend hydroxyapatite beads in 5 ml&nbsp; (10&nbsp; column volumes)&nbsp; of '''buffer A''' 10 mM NaPO , 1 mM EDTA, pH 7.0 sodium phosphate buffer. <br>Let beads settle for 3 min. Carefully decant the liquid and fine particles. 

<br> 

<br> 

3.&nbsp;<strike></strike>Add 50 ml of N5-EF eluate (should contain at least 500 mM&nbsp;NaCl) from AX 10000 column or Qiagen tip 2500 to each 50 ml tube containing HA beads.<br> 

Cap the tubes and tighten the caps. 

Put onto a rotating mixe, mix at room temperature,&nbsp; let plasmid DNA bind to the beads for 10 to 30 min.<br> 

4. Spin down beads, use&nbsp; '''buffer A''' to transfer beads to one Pierce 10 ml spin column. 

<br> 

'''buffer A '''<br> 

<br> 

5. Spin at 1000 rpm for 1 min to remove residual buffer.&nbsp; 

<br> 

6. Elute plasmid DNA with 10 X 1 ml of&nbsp; '''0.4 M '''phosphate buffer, pH 6.8~ 7.0, collect 1 ml fractions:<br> 

<br> 

*6.1 Add 1 ml, soak for 1~2 min, spin at 1000 rpm for 30 sec. 
*6.2 Add 0.5 or 1 ml, soak for 1~2 min, spin at 1000 rpm for 30 sec. 
*6.3 repeat the above elution 6.2 step, check OD260 using nanodrop until OD260 &lt; 50 ug/ul.

<br> 

7. Use nanodrop to measure DNA conceration, OD260/280 ratio, and check purity by agarose gel electrophoresis.<br> 

8. Combine fractions containing plasmid DNA, filter through a 0.22 micron filter in the biosafty hood. label tubes and store them at -20 oC.<br> 

9. Combine used beads and store in cold room. Beads can be regenerated using 1M Phosphate Solution '''for protein purification''' only.<br> 

10/14/2013<br> 

Plasmid DNA purified using hydroxyapatite beads. 0.5 ml per fraction, load 10 ul to gel, concentrations:<br> 

38, 322, 333, 227, 171, 168, 126 ng/ul 

[[Image:Plasmid hydroxyapatide.JPG]]<br> 

<br> 

10/16/2013 

Plasmid DNA purified using hydroxyapatite beads. 0.5 ml per fraction, load 600ng to gel, concentrations: 

170, 209, 218, 305, 642, ng/ul ,OD260/280 ratio:~1.9<br> 

[[Image:Plasmid QC 10-16-2013.JPG]]<br> 

<br> 

<br> 

<br> 

== Buffers <br>  ==

'''1M Phosphate Solution, pH 6.8'''<br> 

In a 1L flask dissolve 119.98g of sodium phosphate monobasic in 1L of&nbsp; ddH2O. <br> 

Prepare a 1M sodium phosphate dibasic solution in a 1L flask by dissolving 141.96g of sodium phosphate dibasic in 1L ddH2O. <br> 

Combine the mono- and di- solutions at a ratio of 1:1. <br> 

Optional: adjust pH to 6.8 by adding sodium hydroxide (to increase pH) or phosphoric acid (to decrease pH).<br><br> 

'''10 mM Phosphate Buffer'''<br> 

<br> 

10 ml of 1M Phosphate Solution, pH 6.8, <br> 

250 ml of 4 M NaCl 

2 ml of 0.5M EDTA. <br> 

Add ddH2O to a volume of 1000 ml. <br> 

Autoclave.<br> 

<br> 

<br>'''0.4 M Phosphate Buffer'''<br> 

400ml of 1M Phosphate Solution, pH 6.8~7.0, <br> 

1 ml of 0.5M EDTA. <br> 

ddH2O to 500 ml<br> 

Autoclave. 

<br> 

'''Plasmid Purification Buffers for AX10000 column'''<br> 

{|
|-
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | Buffer Name<br> 
| bgcolor="#ffffcc" | Recipe<br> 
| bgcolor="#ffffcc" | Buffer Name 
| bgcolor="#ffffcc" | Recipe 
| bgcolor="#ffffcc" | Buffer Name 
| bgcolor="#ffffcc" | Recipe<br> 
| bgcolor="#ffffcc" | Buffer Name 
| bgcolor="#ffffcc" | Recipe
|-
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | &nbsp; '''N2-EF''' 
| bgcolor="#ffffcc" | 1 L 
| bgcolor="#ffffcc" | '''N3-EF''' 
| bgcolor="#ffffcc" | 1 L 
| bgcolor="#ffffcc" | '''N4-EF'''<br> 
| bgcolor="#ffffcc" | 1L<br> 
| bgcolor="#ffffcc" | '''N5-EF''' 
| bgcolor="#ffffcc" | 1L
|-
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | 12.1 g 
| bgcolor="#ffffcc" | 100 mM Tris 
| bgcolor="#ffffcc" | 12.1 g 
| bgcolor="#ffffcc" | 100 mM Tris <br> 
| bgcolor="#ffffcc" | 12.1 g <br> 
| bgcolor="#ffffcc" | 100 mM Tris<br> 
| bgcolor="#ffffcc" | 12.1 g<br>
|-
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | 15&nbsp;% EtOH 
| bgcolor="#ffffcc" | 150 ml 
| bgcolor="#ffffcc" | 15&nbsp;% EtOH 
| bgcolor="#ffffcc" | 150 ml 
| bgcolor="#ffffcc" | <strike></strike>15&nbsp;% EtOH<strike><br></strike> 
| bgcolor="#ffffcc" | 150 ml <br> 
| bgcolor="#ffffcc" | 15&nbsp;% EtOH <br> 
| bgcolor="#ffffcc" | 150 ml <br>
|-
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | 900 mM KCl 
| bgcolor="#ffffcc" | 67.1 g 
| bgcolor="#ffffcc" | 1.15 M KCl 
| bgcolor="#ffffcc" | 85.7 g 
| bgcolor="#ffffcc" | 1.15 M KCl<br> 
| bgcolor="#ffffcc" | 85.73 g<br> 
| bgcolor="#ffffcc" | 1 M KCl 
| bgcolor="#ffffcc" | 74.56 g
|-
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | 0.15&nbsp;% TX 100 
| bgcolor="#ffffcc" | 1.5 g Triton X-100<br> 
| bgcolor="#ffffcc" | 0.15&nbsp;% TX 100 
| bgcolor="#ffffcc" | 1.5 g Triton X-100<br> 
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | <br>
|-
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | pH '''6.3''' with H<sub>3</sub>PO<sub>4</sub><br> 
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | pH '''6.3''' with H<sub>3</sub>PO<sub>4</sub><br> 
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | pH '''6.3''' with H<sub>3</sub>PO<sub>4</sub><br> 
| bgcolor="#ffffcc" | <br> 
| bgcolor="#ffffcc" | pH 8.5 with H3PO4<br>
|}

<br>'''Hydroxyapatite hydration''': Weight out 1g of hydroxyapatite and add to a 50ml tube. Add 6ml of 10 mM phosphate buffer to the hydroxyapatite and invert to mix. Let beads settle down for 10 min and remoe fine particles by decanting.<br> 

Store for extended periods of time at 4°C.<br> 

<br> 

<br> 

<br> 

'''Ref:'''<br> 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230181/<br> 

=  =

10/6/2013. 

1 ml E coli lysate, precipitate with 3 M KCl, adjust to pH 4.0 after adding KCl to final conc 1M. 

or precipitate with 3 M KoAc (P3). 

Bind to 10 mg HA beads. 

HA beads were partially dissolved by KoAc, only 50% of the beads is intact after overnight incubation. 

Miniprep<br> 

[[Image:HA plasmid KCl vs KAc.JPG]] 

Lane 1: Precipitate with KCl 

Lane 2: Precipitate with KoAc 

[[Category:Protocols]]