Paul.xu at 13:40, 18 October 2012

2012-10-18T13:40:50Z

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PEG: quite expensive.

Purity is not good 

 

== Protocol 1: 1 L culture with ~3 to 5 ml QXL 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 '''20''' ml of P3.

6. Add '''15''' ml of '''5 M''' CaCl2 to final conc of 1 M, cool on ice for 5 min.

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

8. Add '''20''' ml of  40% PEG 8000 to  supernatant (1:4, v/v). Transfer to ultracentrifuge tubes, balance to 0.1 g. Number tubes with small piece of adhesive tape. Put into a 35K rotor. 9. Spin for 15 min at 25000 rpm, 20 oC using ultracentrifuge. 

10. Make sure you know where is the pellet as it is transparent. Resuspend "pellet" in 0.8 ml water, wash 5 times with water, transfer to a clean tube. Measure DNA concentration using Nanodrop. Calculate yield. 

11. Add X ul of 10% Triton X-114 (10X) to the plasmid DNA solution to adjust the final concentration of Triton X-114 to 1% (1:10). Mix by votexing. Put on ice.

12. Incubate on ice for 5 to 10 min. You will see the solution is clear.  Aliquot to 1.5 ml tubes. 

13. Incubate at 42~55 oC for 5 to 10 min. You will see the solution is turbid.

14. Spin at 13000 rpm (microcentrifuge, max speed) for 2.5 to 5 min. Carefully collect and combine supernatant to a new tube (Do not take any button phase). Discard the tubes containing bottom phase.

15. Equilibrate 3 to 5 ml Q XL column with QBT or 5 CV of 0.5M KAc (potassium acetate), pH 5.5.

16. Load DNA solution from step 14 to Q XL column, collect FT. The OD260 should be very low (3 ng/ul or none) in FT. 17. Wash with 3 CV (e.g., 3 X3 ml) of 25 mM Tris-HCl, 0.75 M NaCl, 1 mM EDTA, pH 8.0. Collect wash. OD260 should be low.

18. Wash with 5 CV (e.g., 3X 5 ml) of 25 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA, pH 8.0. Collect wash.  DO 260 should be low. 19. Elute DNA with 5 CV (e.g., 15 ml) of 50 mM Tris-HCl, 1 M NaCl, pH 8.0. Collect eluate.

(after elution, clean the column using cleaning solution: 15 ml of 0.5 M NaOH, 2 M NaCl, wash column with water and soak the resin in 20% ethanol until next use. The beads should be stored at 4 oC for long term storage).

20. If fractions are collected, measure each fraction with Nanodrop and combine fractions containing DNA.

21. Add equal volume of  isopropanol, (optionally add 3M potassium acetate, 10% final volume). Transfer to ultracentrifuge tube, balance the tubes with isopropanol. 

22. Spin at 25000 rpm, 20 oC for 15 min to precipitate DNA. 

'''Step 23 and 26 should be peformed inside a biosafety hood'''

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

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

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

26. 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.

26. 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.

27. 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: HiTrap 1 ml or 5 ml QXL column ==

1. Grow high copy number plasmid in DH5alpha in 500 ml or 1L 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 20 ml of P3. 

6. Add 15 ml of 5 M CaCl2 to final conc of 1 M, cool on ice for 5 min. 

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

++++++++++++ 

8. Add 20 ml 40% PEG 8000 to  supernatant (1: 4 v/v) 

 

''Alternatively,'' the DNA in supernatant from step 7 can be precipitated first with isopropanol, dissolve DNA in 1 ml of water, add 0.25 ml of 4 M NaCl (final is 0.8 M), add 0.25 ml of 40% PEG8000 (final is 6.5%) and spin down DNA using a benchtop centrifuge. 

 

9. Spin for 10 min at 25000 rpm, ultracentrifuge. 

10. Resuspend pellet in 1 to 5 ml  '''water''', aliquote to 1.5 ml tubes. 

11. Add '''X ul of 10% Triton X-114 (10X) '''to the plasmid DNA solution to adjust the final concentration of TritonX-114 to '''1%''' '''(1:10).''' Mix by votexing.

12. Incubate '''on ice for 10 min'''. 

13. Incubate at 42~55 oC for 10 min

14. Spin at 13000 rpm (microcentrifuge) for 5 min. Collect and combine '''supernatant''' to a new tube,  chill on ice. Discard the tubes containing bottom phase. 

15. Equilibrated Q XL column with '''QBT '''or 5 CV of  0.5M KAc (potassium acetate), pH5.5

16. Load 1~5 ml '''Supernatant '''(containing plasmid DNA) slowly to '''1 ml Q XL column''' using a syringe. 

 

Note: if a large volume of DNA sample or supernatant is to be applied, use a peristaltic pump at a flow rate of 1 ml/min to load sample. 

 

17. Wash with 3 ml of 25 mM Tris-HCl, 0.75 M NaCl, <strike>'''1% Triton X-100 or 1% Triton X-114,'''</strike> 1 mM EDTA, pH 8.0 by pushing liquid '''slowly '''to the column using  a syringe. 

<strike>18. Wash column with 25 mM Tris-HCl, 1 mM EDTA, pH 8.0 until Triton X-114  is completely washed off column.</strike> 18. Wash with 5 ml of 25 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA, pH 8.0 by pushing liquid '''slowly''' to the column using a syringe. 19. Elute DNA with 3 ml of 50 mM Tris-HCl,  1 M NaCl, pH 8.0 by pushing liquid '''slowly''' to the column using a syringe. 

(after elution, clean the column using cleaning solution: 5 ml of 0.5 M NaOH, 2 M NaCl using a syringe). 

20. Combine fractions containing DNA. 

21. Add 0.7 volumes  of isopropanol,  (optionally add 3M potassium acetate, 10% final volume). 

22. Aliquot to 1.5 ml Eppendorf tubes, Spin at 13000 rpm for 15 min using a benchtop centrifuge to precipitate DNA. 

23. Add 300 ul of 80% ethanol gently, spin briefly. 

 

'''Step 24 and 25 should be peformed inside a biosafety hood''' 

 

24. Remove ethanol, and then dry inside a biosafety hood. 

25. Add 0.3 ml sterilized TE to dissolve DNA, add 3 X 100 ul to wash DNA off the tubes. Combine and take 5 ul out to another tube. 

 

26. Measure DNA concentration and purity using a Nanodrop; Run gel to check RNA level; Digested with restriction enzymes to confirm identity. 

27. Analyse endotoxin level to see if it is acceptable for transfection.

 

== '''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, Column: Plasmid Select, FineLINE 70, 14 cm bed height (CV = 566 ml) Buffer A: 2 M (NH4)2SO4, 10 mM EDTA, 100 mM Tris-HCI, pH 7.0 

Buffer B: 1.4 M NaCl, 2 M (NH4)2SO4, 10 mM EDTA, 100 mM Tris-HCI, pH 7.0 Flow velocity: 120 cm/h 

 

3. 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 

 

== 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).

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 PEG Q XL - Revision history

Paul.xu at 13:40, 18 October 2012