Editing Plasmid MaxiPrep HIC PLasmidSelect (section)

== Hexyl 650C (Toso) + PlasmidSelect Protocol  ==

Hexyl 650C can be used to remove endotoxin at 2 to 2.2M ammonium sulfate. Plasmid will not bind to the resin.Plasmid will bind at 3 M ammonium sulfate.<br> 

The problem of this protocol is:<br> 

both Hexyl 650C and PlasmidSelect resin become very dirty after loading sample. Elute come off PlasmidSelect resin show brown color. OD260/280 ratio is not good.<br>

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1. For high copy number plasmid, spin down 600 (OD600=2) to 1000 ml (OD600=1.5) overnight cultured cells.<br> 

2. Add 20 ml of P1 (50 mM Tris-HCl, 10 mM EDTA pH8.0) with RNase. 

3. Add 20 ml of P2 (200 mm NaOH, 1% SDS solution), lyse for about 5 min. 

4. Add 20 ml of P3 (3 M potassium acetate, pH ~ 5. Dissolve 294.5 g potassium acetate in 500 ml dH2O. Adjust the pH to 5.5 with ~110 ml glacial acetic acid and bring to 1 L with ddH2O).<br> 

&nbsp;5. Put on ice for 10 min, spin at 6000 rpm&nbsp; for 10 min. Filter floating stuff with coffer filter. 

6. Collect supernatant, add 3.75M ammonium sulphate to final concentration of 2.0M. Spin again if you see precipitate. 

7. Load to a column packed with 6 ml Hexyl 650C (Toso) preequilibrated with 2 CV of '''Buffer A''': <br> 

*2 M (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> ammonium sulfate (AS) in 50mM Tris-HCl, 10 mM EDTA (TE) buffer, pH 7.5.&nbsp; <br>

C'''ollect flow through.''' 

8. Wash column with 5CV of Buffer A. 

9. Load solution from step 7 and 8 to 2 to 5 ml of PlasmidSelect column (preequilibrated with 2 CV of Buffer A). '''Do not discard flow through.'''<br> 

10. Wash column with 5 to 10 CV of Buffer A.<br> 

11. Elute DNA with 3 to 5 CV '''Buffer B:'''<br> 

*1 M NaCl, 1.7 M (NH4)2SO4, 10 mM EDTA, 50 mM Tris-HCl, pH 7.5.<br>

Collect&nbsp; 1 ml fractions, check each fraction for DNA concentration using nanodrop, combine fractions containing DNA.<br> 

12. Add 0.7 volume isopropanol. Put on ice for 10 min or cold room overniht and spin at 12000 rpm for 15 min to precipitate DNA.<br> 

13. Wash with 70% ethanol spay. Dry inside a biosafety hood. Dissolve DNA in 3 X 200 to 5 X 200 ul TE. <br> 

14. Determine DNA purity using nanodrop (OD260/280 ratio), run 1 ul on 1% gel, and check DNA identity by restriction digestion.<br> 

15. Upload data to ELN.<br> 

16. Regenerate Hexyl 650C<br> 

*10 CV water, 2 CV 0.1N NaOH, then 10 CV water, store resin in 20% ethanol.<br>

17. Regenerate PlasmidSelect:<br> 

*5 CV 0.1N NaOH/1.5M NaCl, then 10 CV water, store resin in 20% ethanol.<br>

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[[Category:Protocols]]