Paul.xu at 13:39, 18 October 2012

2012-10-18T13:39:48Z

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Problem: Low yield using CTAB precipitation method. 

== Protocol 1 ==

1. Inoculate 500 mL 2xYT or LB/Amp or Kan from 1-5 mL overnight

2. Grow at 37 ºC overnight.

3. Spin cells at 6000 rpm for 10 min at 4ºC using GS3 rotor.

4. Resuspend cells in 8 mL P1.

5. Add 8 mL P2. Stir very gently using a long 5 ml plastic pippette, until homogenous and clear. Put on ice for 10 min.

6. Add 10 mL P3 (3 M KAc, pH 5), or S3 (Qiagen), mix, put on ice for 10 min. S3 is the best.

7. Spin 6000 rpm at 4ºC for 10 min.

8. Filter supernatant using autoclaved cheesecloth or coffee filter paper.

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Optional steps

9. Add 3% CTAB solution to supernatant to final concentration of 0.3%.  <strike>0.18%</strike>

10. Spin down CTAB-DNA complex. Dissolve pellet in 0.7 M NaCl or 3M KAc (Potassium Acetate)

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11. Load DNA solution to Q Sepharose FF column or HiTrap Q FF column (GE Healthcare) pre-equilibrated with QBT

12. Wash column with QC buffer (0.27~0.45M NaCl).

13. Elute DNA with QF buffer.

14. Precipitate DNA using 0.6 vol of isopropanol or 2 vol of ethanol.

15. Wash DNA precipitate with 75% ethanol.

16. Dry DNA in biosafety hood.

17. Dissolve DNA in water or TE.

18. Analyse DNA purity by agarose gel and nanodrop.

'''Regenerate Q column:'''

19. Add 1 mg/mL DNase I, 50mM Tris-HCl, 10mM MgCl2, 250mM NaCl, pH 7.6 and put column or resin in 37 oC incubator over night.

 20. Wash with: 5CV 1M NaOH 5CV H2O 5 CV isopropanol 5 CV water 5 CV 1M phosphoric acid 5 CV water 5 CV 2M NaCl. 5 CV 0.35 M NaCl in 20% ethanol.

'''Buffer QBT'''

Dissolve 43.83 g NaCl, 10.46 g MOPS (free acid) in 800 ml dH2O. Adjust the pH to 7.0 with NaOH. Add 150 ml pure isopropanol and 15 ml 10% Triton X-100 solution (v/v). Adjust the volume to 1 liter with dH2O.

750 mM NaCl 50 mM MOPS pH 7.0 15% isopropanol (v/v) 0.15% Triton® X-100 (v/v)

'''Buffer QC'''

Dissolve 58.44 g NaCl and 10.46 g MOPS (free acid) in 800 ml dH2O. Adjust the pH to 7.0 with NaOH. Add 150 ml pure isopropanol. Adjust the volume to 1 liter with dH2O.

1.0 M NaCl 50 mM MOPS pH 7.0 15% isopropanol (v/v)

 '''Buffer QF'''

Dissolve 73.05 g NaCl and 6.06 g Tris base in 800 ml dH2O and adjust the pH to 8.5 with HCl. Add 150 ml pure isopropanol. Adjust the volume to 1 liter with dH2O.

1.25 M NaCl 50 mM Tris·Cl pH 8.5 15% isopropanol (v/v) 

== Protocol 2 ==

ref: http://www.freepatentsonline.com/7285651.html 

1. Spin down cells 

2. Add STET buffer (8% Sucrose, 5% Triton X-100, 50mM EDTA, 50mM Tris pH 8.0) at  1:10 ratio. 

3. Resuspend cells 

4. Lyse cells  by incubating   with Ready-Lyse lysozyme (Epicentre) at 500 U/mL for 1 hour at 37° C

5. Heat suspenion at 70° C, cool to room temp 

6. Add Celpure P300 at 4 g/100ml 

7. Filter to remove junk 

8. Add 3% (w/v) CTAB in 40 mM NaCl to final conc of 0.3% w/v CTAB. 

9. Centrifuge to precipitate DNA 

10. Dissolve DNA with 0.75 M NaCl. 

11. Add absolute ethanol slowly to DNA solution to  final ethanol concentration of 50% v/v. 

12. Spin to precipitate DNA 

13. Wash pellet with 90% ethanol. 

14. Air dry inside a biosafety hood. 

15. Dissolve DNA in TE. 

[[Category:Protocols]]

Plasmid MaxiPrep by CTAB Q Sepharose FF - Revision history

Paul.xu at 13:39, 18 October 2012