Editing Plasmid MaxiPrep Hydroxyapatite (section)

== 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. 

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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. 

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'''buffer A '''<br> 

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

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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> 

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*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.

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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> 

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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> 

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