Paul.xu at 13:54, 31 October 2012

2012-10-31T13:54:31Z

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== Protocol 1: 1 L culture with 1 or 5 ml  Q XL column ==

For 4 L culture a column packed with 5 ml QXL HiTrap should be used. 

For 1 L culture, the column can be packed with 1 ml of QXL HiTrap.

1. Grow high copy number plasmid in DH5alpha in 1 L LB/Amp or LB/Kan o/n at 37oC.

2. Spin down cells

3. Add 15 ml P1 buffer ('''with RNase A''').

4. Lyse cells with '''15 '''ml of P2.

5. Neutralize with '''15''' ml of P3.

6. Spin at 12000 rpm  for 10 min or 6000 rpm for 15 min, high speed centrifuge, collect supernatant, filter liquid using a coffee filter.

7. Precipitate DNA with 0.7 volume of isopropanol, incubate on ice for 30 min, spin at 12000 rpm for 15 min. 

8. Dissolve DNA in 5 X 1 ml of Buffer EQ: (0.3 M NaCl, 25 mM NaOAc, pH 5.0) or Buffer B. 

9. Spin at 13000 rpm for 5 min to pellet insoluble matter, collect and combine supernatant. 

10. Equilibrate 5 ml HiTrap Q XL column with 5 CV of Buffer EQ: (0.3 M NaCl, 25 mM NaOAc, pH 5.0) or Buffer B. 

11. Load DNA solution (supernatant from step 9) to 5 ml HiTrap Q XL column using a syringe or a peristaltic pump.

12. Wash with '''3''' CV (e.g., 3 X3 ml) of '''Buffer A''' (25 mM Tris-HCl, '''0.75 M '''NaCl, 10% EtOH, 0.5% Triton X-114, 1 mM EDTA, pH 8.0). This wash contains Triton X-114, which has high UV absorbance, so there is no need to measure OD260.

13. Wash with '''5 to 15''' CV (e.g., 3X 5 ml) of '''Buffer B''' (25 mM Tris-HCl, '''0.5 M''' NaCl, 1 mM EDTA, pH 8.0). Collect wash.  Check OD260 in the last few drops.

 14. Elute plasmid with 10 CV  of linear gradient using '''filtered Buffer B''' to filtered '''Buffer C''' (25 mM Tris-HCl, '''1.6 M''' NaCl, 1 mM EDTA, pH 8). Collect 1 ml fractions. 

*DNA can be also eluted with 50 mM Sodium Phosphate, 1 M NaCl , pH 9.6. 
*After elution, clean the column with  '''filtered CIP''' cleaning solution: 15 ml of '''0.5 M NaOH, 2 M NaCl''' using a syringe, wash column with water and soak the resin in 20% ethanol until next use. The column should be stored at 4 oC for long term storage.

15. Combine fractions containing plasmid DNA.

16. Add 0.7 X volume of  isopropanol.  Transfer to ultracentrifuge tube, balance the tubes with isopropanol. Incubate on ice for 30 min. 

17. Spin at 25000 rpm, 20 oC for 15 min to precipitate DNA. (You can also spin down plasmid at 12000 rpm for 20 min). 

'''Step 18 and 21 should be peformed inside a biosafety hood'''

19. Take centrifuge tubes to the biosafety hood. Discard liquid careful, make sure you know where is the pellet as it is invisible (transparent).

20. Spray some  80% ethanol into the tube gently, do not disturb pellet. 

21. Remove ethanol gently,  dry pellet for 5 min inside a biosafety hood.

22. Add 200 ul sterilized TE to dissolve DNA (you will not see pellet because it is transparent), add 3 X 200 ul to wash DNA off the tubes. Combine and take 10 ul out to an Eppendorf tube.

23. Take 2 ul to measure DNA concentration and purity using a Nanodrop; Run 2 ul on gel to check RNA level; Digest with restriction enzymes and run on agarose gel to confirm identity.

24. Optional: Analyse endotoxin level to see if it is acceptable for transfection.

Yield: should be ~1 mg/L culture Quality: RNA <1%, 260/280: >1.8, mostly supercoil DNA 

== Protocol 2: 1 L culture with 1 to 3 ml  Q XL column ==

For 4 L culture a column packed with 10 ml QXL resinshould be used. 

For 1 L culture, the column can be packed with 3 to 5 ml of QXL resin.

1. Grow high copy number plasmid in DH5alpha in 1 L LB/Amp or LB/Kan o/n at 37oC.

2. Spin down cells

3. Resuspend cells with '''20''' ml P1 buffer ('''no RNase''').

4. Lyse cells with '''20 '''ml of P2.

5. Neutralize with '''10''' ml of P3.

6. Add '''2  '''ml of '''5 M''' CaCl2 to final conc of 0.2 M, let Ca2++ to bind RNA for 15 min at room temperature.

7. Spin at 12000 rpm for 10 min, high speed centrifuge, collect supernatant, filter liquid using a coffee filter.

8. Load to Hexyl 650C column (5 ml resin, equilibrated with '''0.5 X''' '''P3, 0.5 X diluted P3'''). Wash with 52 ml water. '''Collect flow through.''' 

9. Immediately after water wash, regenerate Hexyl 650C column with 3 CV of 0.1 M NaCl and then with 10 ~15 CV of water. Store column in 20% ethanol.

10. Equilibrate 5 ml HiTrap Q XL column with 5 CV of Buffer EQ: (0.3 M NaCl, 25 mM NaOAc, pH 5.0) or Buffer B. 

11. Load flow through from step 8 to 5 ml HiTrap Q XL column using a peristaltic pump.

12. Wash with '''3''' CV (e.g., 3 X3 ml) of '''Buffer A''' (25 mM Tris-HCl, '''0.75 M '''NaCl, 10% EtOH, 0.5% Triton X-114, 1 mM EDTA, pH 8.0). This wash contains Triton X-114, which has high UV absorbance, so there is no need to measure OD260.

13. Wash with '''5 to 15''' CV (e.g., 3X 5 ml) of '''Buffer B''' (25 mM Tris-HCl, '''0.5 M''' NaCl, 1 mM EDTA, pH 8.0). Collect wash.  Check OD260 in the last few drops.

 14. Elute plasmid with 10 CV  of linear gradient using '''filtered Buffer B''' to filtered '''Buffer C''' (25 mM Tris-HCl, '''1.6 M''' NaCl, 1 mM EDTA, pH 8). Collect 1 ml fractions. 

*DNA can be also eluted with 50 mM Sodium Phosphate, 1 M NaCl , pH 9.6. 
*After elution, clean the column with  '''filtered CIP''' cleaning solution: 15 ml of '''0.5 M NaOH, 2 M NaCl''' using a syringe, wash column with water and soak the resin in 20% ethanol until next use. The column should be stored at 4 oC for long term storage.

15. Combine fractions containing plasmid DNA.

16. Add 0.7 X volume of  isopropanol.  Transfer to ultracentrifuge tube, balance the tubes with isopropanol. Incubate on ice for 30 min. 

17. Spin at 25000 rpm, 20 oC for 15 min to precipitate DNA. (You can also spin down plasmid at 12000 rpm for 20 min). 

'''Step 18 and 21 should be peformed inside a biosafety hood'''

19. Take centrifuge tubes to the biosafety hood. Discard liquid careful, make sure you know where is the pellet as it is invisible (transparent).

20. Spray some  80% ethanol into the tube gently, do not disturb pellet. 

21. Remove ethanol gently,  dry pellet for 5 min inside a biosafety hood.

22. Add 200 ul sterilized TE to dissolve DNA (you will not see pellet because it is transparent), add 3 X 200 ul to wash DNA off the tubes. Combine and take 10 ul out to an Eppendorf tube.

23. Take 2 ul to measure DNA concentration and purity using a Nanodrop; Run 2 ul on gel to check RNA level; Digest with restriction enzymes and run on agarose gel to confirm identity.

24. Optional: Analyse endotoxin level to see if it is acceptable for transfection.

Yield: should be ~1 mg/L culture Quality: RNA <1%, 260/280: >1.8, mostly supercoil DNA 

10/20/2012 

5 ml Q-XL column, load DNA solution from 1 L culture [[Image:Q xl 10 20 2012.jpg]] 

 

{| width="487" cellspacing="1" cellpadding="1" border="1"
|-
| 
| 1 
| 2 
| 3 
| 4 
| 5 
| 6 
| 7 
|-
| 
|
FT,

10 ml 

|
Buffer A wash,

12 ml 

| Buffer B wash, 17 ml 
| Buffer B wash, 33 ml 
|
Elute

fraction 1, 2 ml 

| Elute
fraction 3, 2 ml 

| Elute
fraction 5, 2 ml 

|-
| Load to gel 
| 5 ul 
| 5 ul 
| 5 ul 
| 5 ul 
| 2 ul 
| 2 ul 
| 2 ul 
|}

Can not completely wash out RNA with Buffer B (25 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA, pH 8.0) 

== '''Other columns:''' ==

QIAGENO®, DEAE Sepharose®, Q Sepharose, DEAE Sephadex®, Poros 20 M/P and/or Poros 50 M/P 

Qiagen column: anion exchanger (70-100 μm particle size, 2.5 μmol DEAE/g of chromatographic material) 

== '''Optional protocol:''' ==

1. Column: RESOURCE Q, 6 ml (prepacked with SOURCE 15 Q) Sample: from step 16, dilute to 0.4 M NaCl (2.5 folds) Sample load: 1 to 140 ml Sample conductivity: 38 mS/cm Buffer A: 25 mM Tris-HCl, 1 mm EDTA, pH 8.0 Buffer B : 25 mM Tris-HCl, 1 mM EDTA, 1.0 M NaCl, pH 8.0 Flow velocity: 300 cm/hr Gradient: 40 -100%B, 20 CV Then precipitate DNA as step 17 to 21. 

 

2. SOURCE 30Q, FineLINE 35, 12 cm bed height (CV = 137 ml) Buffer A: 0.4M NaCl, 10 mM EDTA, 100 mM Tris-HCI, pH 7.0 

Buffer B: 1.0M NaCl, 10 mM EDTA, 100 mM Tris-HCI, pH 7.0 Flow velocity: 120 cm/h 3. SOURCE 15Q Column size: 6 ml, 16 mm i.d. x 30 mm (RESOURCE Q® from Pharmacia Biotech) Chromatography System: ÄKTA explorer Equilibration Buffer A: 25 mM Tris-Cl, 1 mM EDTA, pH 8.0 Buffer B with high salt: 25 mM Tris-Cl, 1 mM EDTA, 1.0 M NaCl, pH 8.0 Buffer for cleaning in place: 2 M NaCl, 0.5 M NaOH Gradient: 40-100% B, 20 CV Flow velocity: 150-900 cm/hr The pooled fractions from the first chromatography step are diluted and the conductivity is adjusted to 38 mS/cm. 140 ml of the sample are applied to the column pre equilibrated with buffer A. The plasmid is eluted using a linear gradient of 40 - 100 % buffer B. The fractions were collected. 

 

== Buffers ==

 

'''P1 ''' 

<strike>10 μg/ml RNase A</strike>, 50 mM Tris-HCl, pH8.0, 10 mM EDTA; 

'''P2''' 

200 mM NaOH, 1% SDS 

'''P3''' 

3 M KAc, pH 5.5 

'''QBT''' 

750 mM NaCl, 50 mM MOPS, 15% isopropanol or ethanol, pH 7.0, 0.15% Triton X 100 

'''QC''' 

1.0 M NaCl, 50 mM MOPS, 15% isopropanol or ethanol, pH 7.0 

'''QN''' 

1.6 M NaCl, 50 mM MOPS, 15% isopropanol or ethanol, pH 7.0. 

'''Endotoxin Removal Buffer''' 

750 mM NaCl, 10% Triton X 100, 50 mM MOPS, pH 7.0 

'''QF''' 

1.25M NaCl, 50 mM Tris-HCl, 15% ethanol, pH 8.5 

'''TE''' 

10 mM Tris-HCl, 1mM EDTA, pH 8.0 

 

== '''Optional method to further reduce endotoxin level''' ==

Dissolve DNA in water and add Triton X-114 to a final concentration of 0.1-1%. 

Incubate the solution on ice, mix occasionally, for 30 min. 

Heat the solution to 42 oC for 5 min. 

Spin at 20,000× g for 5 min or filter. 

Add 0.7 volumes of isopropanol to supernatant

Spin for 10 to 15 min to precipitate DNA.

Wash DNA with 80% ethanol

Dry the resulting pellet in a biosafety hood and resuspend in TE. 

 

== Purification of Plasmid using DEAE Q Sepharose ==

1. P1, P2, P3. 

2. Add 0.7 vol of isopropanol to precipitate DNA. 

3. Dissolve DNA in water and load onto a DEAE Q Sepharose column with a bed volume of 2 ml. 

4. Elute DNA is eluted with a NaCl gradient of 

Buffer A: 10 mM Tris/HCl, 1 mM EDTA, 0.75 M NaCl, pH 8.0;

Buffer B: 10 mM Tris/HCl, 1 mM EDTA, 1.5 M NaCl, pH 8.0;

5. Precipitate DNA with ethanol and resuspended in TE. 

 

== Purification of Plasmid using  Nucleobond® EF 500 cartridge ==

Equilibrate column with 5 ml equilibration buffer ('''N2-EF''', 100 mM TRIS/phosphate, pH 6.3, 15% ethanol, 900 mM KCl, 0.5% Triton X-114). 

Apply clarified (by filtration) bacterial lysate  onto the cartridge.

Wash 4×12 ml buffer '''N3-EF '''(100 mM TRIS/phosphate, pH 6.3, 15% ethanol, 1150 mM KCl, 0.5% Triton X-114).

4×12 ml N4-EF 

'''N4-EF''' (I guess): Wash Buffer (Store at RT)

100 mM Tris 15% EtOH 1.15 M KCl Phosphoric Acid: added until pH is 6.3 

 

Elute by adding elution buffer '''N5-EF''' (100 mM TRIS/phosphate, pH 7.0, 15% ethanol, 1500 mM KCl).

Precipitate DNA by adding 0.7-0.8% isopropanol and centrifuging (30 min >15,000 G),

Wash with endotoxin-free 70% ethanol precooled to 4° C.

Dry and resuspend in endotoxin-free TE buffer. 

[[Category:Protocols]]

Plasmid MaxiPrep by Ca Q XL - Revision history

Paul.xu at 13:54, 31 October 2012