Paul.xu at 13:38, 18 October 2012

2012-10-18T13:38:37Z

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== 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. 

The problem of this protocol is: 

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. 

 

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

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). 

 5. Put on ice for 10 min, spin at 6000 rpm  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''': 

*2 M (NH4)2SO4 ammonium sulfate (AS) in 50mM Tris-HCl, 10 mM EDTA (TE) buffer, pH 7.5.  

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.''' 

10. Wash column with 5 to 10 CV of Buffer A. 

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

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

Collect  1 ml fractions, check each fraction for DNA concentration using nanodrop, combine fractions containing DNA. 

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. 

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

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

15. Upload data to ELN. 

16. Regenerate Hexyl 650C 

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

17. Regenerate PlasmidSelect: 

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

 

 

[[Category:Protocols]]

Plasmid MaxiPrep HIC PLasmidSelect - Revision history

Paul.xu at 13:38, 18 October 2012