Editing Plasmid MaxiPrep by PEG Q XL

PEG: quite expensive.

Purity is not good <br>

<br> 

== 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.<br> 

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 37<sup>o</sup>C. 

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&nbsp;'''15''' ml of '''5 M''' CaCl<sub>2</sub> 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&nbsp; 40% PEG 8000 to&nbsp; 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.<br>9. Spin for 15 min at 25000 rpm, 20 oC using ultracentrifuge. <br> 

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.<br> 

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.&nbsp; Aliquot to 1.5 ml tubes.<br> 

13. Incubate at 42~55 <sup>o</sup>C 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&nbsp;Q XL column, collect FT. The OD260 should be very low (3 ng/ul or none) in FT.<br>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.&nbsp; DO 260 should be low.<br>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&nbsp; isopropanol, (optionally add 3M potassium acetate, 10% final volume). Transfer to ultracentrifuge tube, balance the tubes with isopropanol.<br> 

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

'''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&nbsp; 80% ethanol into the tube gently, do not disturb pellet. <br> 

25. Remove ethanol gently,&nbsp; 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<br>Quality: RNA &lt;1%, 260/280: &gt;1.8, mostly supercoil DNA<br> 

== Protocol 2: HiTrap 1 ml or 5 ml QXL column<br>  ==

1. Grow high copy number plasmid in DH5alpha in 500 ml or 1L LB/amp or LB/Kan o/n at 37oC.<br> 

2. Spin down cells<br> 

3. Resuspend cells with 20 ml P1 buffer (no RNase).<br> 

4. Lyse cells with 20 ml of P2.<br> 

5. Neutralize with 20 ml of P3.<br> 

6. Add 15 ml of 5 M CaCl<sub>2</sub> to final conc of 1 M, cool on ice for 5 min.<br> 

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

++++++++++++<br> 

8. Add 20 ml 40% PEG 8000 to&nbsp; supernatant (1: 4 v/v)<br> 

<br> 

''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.<br> 

<br> 

9. Spin for 10 min at 25000 rpm, ultracentrifuge. <br> 

10. Resuspend pellet in 1 to 5 ml&nbsp; '''water''', aliquote to 1.5 ml tubes.<br> 

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

12. Incubate '''on ice for 10 min'''.<br> 

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,&nbsp; chill on ice. Discard the tubes containing bottom phase.<br><br> 

15. Equilibrated Q XL column with '''QBT '''or 5 CV of&nbsp; 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.<br> 

<br> 

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.<br> 

<br> 

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 '''<u>slowly</u> '''to the column using&nbsp; a syringe.<br> 

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

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

20. Combine fractions containing DNA.<br> 

21. Add 0.7 volumes&nbsp; of isopropanol, &nbsp;(optionally add 3M potassium acetate, 10% final volume).<br> 

22. <font color="red">Aliquot to 1.5 ml Eppendorf tubes</font>, Spin at 13000 rpm for 15 min using a benchtop centrifuge to precipitate DNA.<br> 

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

<br> 

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

<br> 

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

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.<br> 

<br> 

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

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

<br> 

== '''Other columns:'''<br>  ==

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

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

== '''Optional protocol:'''<br>  ==

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

<br> 

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

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

<br> 

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

Buffer B: 1.0M NaCl, 10 mM EDTA, 100 mM Tris-HCI, pH 7.0<br>Flow velocity: 120 cm/h<br><br><br> 

<br> 

== Buffers<br>  ==

<br> 

'''P1 '''<br> 

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

'''P2'''<br> 

200 mM NaOH, 1% SDS<br> 

'''P3''' <br> 

3 M KAc, pH 5.5 <br> 

'''QBT''' <br> 

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

'''QC''' <br> 

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

'''QN''' <br> 

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

'''Endotoxin Removal Buffer'''<br> 

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

'''QF'''<br> 

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

'''TE'''<br> 

10 mM Tris-HCl, 1mM EDTA, pH 8.0 <br> 

<br> 

== '''Optional method to further reduce endotoxin level'''<br>  ==

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

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

Heat the solution to 42 oC for 5 min.<br> 

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

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. <br> 

<br> 

== Purification of Plasmid using DEAE Q Sepharose  ==

1. P1, P2, P3.<br> 

2. Add 0.7 vol of isopropanol to precipitate DNA.<br> 

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

4. Elute DNA is eluted with a NaCl gradient of <br> 

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.<br> 

<br> 

== Purification of Plasmid using&nbsp; Nucleobond® EF 500 cartridge <br>  ==

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).<br> 

Apply clarified (by filtration) bacterial lysate&nbsp; 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 &gt;15,000 G), 

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

Dry and resuspend in endotoxin-free TE buffer.<br> 

[[Category:Protocols]]