Plasmid MaxiPrep HIC

For 500 ml ~ 1 L culture

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 for 5 min.

5. Neutralize with 20 ml of P3.

6. Cool on ice for 5 min.

7. Spin at 6000 rpm for 10 min, high speed centrifuge, collect supernatant, filter liquid using coffee filter if necessary.

E. Coli genomic DNA and RNA were precipitated by addition of solid ammonium sulfate at 80% by weight saturation. The pH of the solution was maintained at 5.2+/−0.2. The suspension was allowed to stir slowly overnight at 4° C. The resulting precipitate (containing the genomic DNA and RNA) was; removed by centrifugation at 10,000× g for 30 min.

Chromatography

Hexyl 650C (Toso)

Hexyl 650C can be used to remove endotoxin at 2.2M ammonium sulfate. Plasmid will not bind to the resin.

at 3 M ammonium sulfate plasmid will bind to resin and can be eluted using 1 M ammonium sulfate.

1. Spin down 500 ml cells

2. Add 20 ml of P1 (50 mM Tris-HCl, 10 mM EDTA pH8.0) without 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. Add 15 ml of 5 M CaCl2. Final conc is 1 M. (This step should be ommitted if P1 has RNase)

1*(60+X)=5*X; (5X-1X)=60; 4X=60;

5. Put on ice for 10 min, spin at 6000 rpm for 10 min. Filter floating stuff with coffer filter.

6. Collect supernatant, add isopropanol to final 70% (adding 2.1 ml isopropanol per 3 ml of clarified lysate). 

7. Spin.

8. Wash pellet with 70% ethanol.

9. Dry and dissolve DNA in TE buffer (10 mM Tris-HCl, 0.1mM EDTA, pH 8.0.) or water, The DNA solution can be frozen at -20.degree. C. until use

10. Pack a Butyl 650S or 650M column with a bed volume of 3 to 5 ml (1.6 cm X 10 cm).

11. Equilibrate column with 2 CV of  3 M ammonium sulfate (AS) in 50mM Tris-HCl, 10 mM EDTA (TE) buffer, pH 7.4

12.  Load solution to column at 1 ml/min

13. Wash column with 15  to 20  ml of  3 M AS in TE buffer, then 15 ml of 80% Ethanol or PE.

14. Elute DNA with 2 ml  of TE or water. (Alternatively collect 1 ml fractions). Discard the first 2 ml.

15. Measure DNA concentration and 260/280 ratio in each fraction. Combine fractions containing DNA.

16. If purity is not good, purify further using Q XL resin.

1. Dissolve pellet in 20mM KPB (potassium phosphate buffer, 20 mM potassium phosphate monobasicsolution combined with 20 mM potassium phosphate dibasic in a proportion to obtain a pH of 6.8), pH 6.8 and 2M ammonium sulfate (AS).

2. Load solution to 1.6 cm X 4 cm (8 ml cv) Hexyl 650C column equilibrated with KPB, pH 6.8, containing 2M AS, at a flow rate of 2 ml/min.

3. Wash column with KPB containing 2M AS.

4. Elute DNA with TE.

5. Clean column with 0.5N NaOH.

6. Wash column with 15 CV of ddH2O.

Butyl 650S

1. Dissolve pellet in TE buffer, pH 7.4, containing 2M AS and 2M ammonium sulfate (AS).

2. Load solution to 1.6 cm X 4 cm (8 ml cv) Hexyl 650C column equilibrated with TE containing 2M  AS, at a flow rate of 2 ml/min.

3. Wash column with TE containing 2M AS.

4. Elute DNA with TE.

5. Clean column with 0.5N NaOH.

6. Wash column with 15 CV of ddH₂O equilibrated with

Butyl 650S to Purify SC Plasmid DNA

Method 1

1. Equilibrate column with 3M ammonium sulfate in Tris-EDTA buffer pH 7.4.

2. Load sample in the Tris-EDTA buffer, pH7.4 w/3 M AS to column.

3. Wash column with 2-3 column volumes of a 3M AS in TE buffer.

4. Elute DNA with a gradient of ammonium sulfate concentration from 3M to 1M over 6 CV.

the first peak contains the relaxed form of the plasmid DNA

the second peak containing the supercoiled form of plasmid DNA

Step gradient:

2.4M AS: relaxed form of the plasmid DNA
1M AS: supercoiled form of plasmid DNA

Method 2

1. Dissovle sample in 50 mM Tris 10 mM EDTA pH 7.4 buffer with 2M NaCl.

2. Load to Butyl  l650S  1 cm X 20 cm column with ~  10 ml bed volume  equilibrated with TE containing 2M NaCl.

3. Was with TE buffer containing 2M ammonium sulfate

4. Elute DNA with TE.

Butyl 650S or 650M to Purify Plasmid DNA

1. Spin down 500 ml cells

2. Add 20 ml of P1 (50 mM Tris-HCl, 10 mM EDTA pH8.0) without 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. Add 15 ml of 5 M CaCl_2. Final conc is 1 M.

1*(60+X)=5*X; (5X-1X)=60; 4X=60; 

5. Put on ice for 10 min, spin at 6000 rpm for 10 min. Filter floating stuff with coffer filter.

6. Collect supernatant, add isopropanol to final 70% (adding 2.1 ml isopropanol per 3 ml of clarified lysate).

7. Spin.

8. Wash pellet with 70% ethanol.

9. Dry and dissolve DNA in TE buffer (10 mM Tris-HCl, 0.1mM EDTA, pH 8.0.) or water, The DNA solution can be frozen at -20.degree. C. until use

11. Pack a Butyl 650S or 650M column with a bed volume of 20 3 to 5 ml (1.6 cm X 10 cm).

12. Equilibrate column with 2 CV of 3 M NaCl 2.2M ammonium sulfate (AS) in 50mM Tris-HCl, 10 mM EDTA (TE) buffer, pH 7.4.

13. Add ammonium sulfate to final conc of 2.2 M. and dilute with 2.2 M AS in Tris-EDTA, pH 7.4 to 10 ml.  Add 5 ml of 3.75 M AS to DNA solution.  Add 75 ml of 5 M NaCl to DNA solution.

14. Load to column at 1 ml/min

15. Wash column with 15  35 ml of 3M NaCl 2.2M AS in TE buffer., then 15 ml of 80% Ethanol or PE.

16. Wash column with 55 ml of 1M AS in TE buffer, pH 7.4,  collect two fractions: 10 ml and 45 ml.

16. Elute DNA with 2 ml then 15  50 ml of  TE or water.  (Alternatively collect 1 ml fractions). Discard the first 2 ml.

17. Measure DNA concentration and 260/280 ratio in each fraction. Combine fractions containing DNA.

18. If purity is not good, purify further using Q XL resin.

9/9/2012

Purify DNA using Butyl 650M

1. Plasmid purified with silica miniprep spin column, using P1 with RNase, and N3

2. Plasmid purified using 650M, add CaCl2 (1 M).

3. Plasmid purified from FT of #2, using silica miniprep spin column

4. Plasmid purified using 650M, add CaCl2 (1 M), did not add NaCl at step 13.

5. Plasmid purified from FT of #4 using silica miniprep spin column.

6. Plasmid purified using 650M, add 60 ml 3M ammonium sulfate at step 5.

7. Plasmid purified with silica miniprep spin column, with P1 without RNase, and P3.

Load 10 ul.

	1	2	3	4	5	6	7
OD260	45.8	64.6	8.9	8.7	3.2	580	157
260/280	1.96	1.85	2.01	1.48	-	2.1	2.08

9/9/2012 Purify DNA using CaCl2 + PEG or Isopropanol Precipitation

DNA from 3 ml culture. + 250 ul P1 with or without RNase, 250 ul P2, 250 ul P3 or N3

+ CaCl2 to 1 M or without CaCl2, Spin, Get supernatant, precipitate with either 10% (final) PEG 8000 or Isopropano. remove liquid, rinse with 200 ul PE, spin brieftly, remove liquid, dry, add 50 ul water. Take 10 ul to load on gel.

8: P1 no RNase, P3 + CaCl₂, then ppt DNA using 10% PEG

9: P1 no RNase, P3 + CaCl₂, then ppt DNA using 10% PEG

10: P1 no RNase, P3 + CaCl₂, then ppt DNA using 10% PEG

11: P1 w RNase, P3, then ppt DNA using 10% PEG

12: P1 w RNase, P3, then ppt DNA using 10% PEG

13: P1 w RNase, N3, then ppt DNA using 10% PEG

14: P1 w RNase, P3, then ppt DNA using isopropanol

15: P1 w RNase, N3, then ppt DNA using isopropanol

16: P1 no RNase, P3+ CaCl₂, then ppt DNA using isopropanol

17: P1 no RNase, P3+ CaCl₂, then ppt DNA using isopropanol

	8	9	10	11	12	13	14	15	16	17
OD260 ng/ul	24.7	17.5	17.6	118.7	133.3	102	87.8	975	504	157
260/280	1.82	1.87	1.88	1.9	1.86	1.91	1.99	1.96	1.97	2.04

Conclusion:

• P1 without RNase + P2 + 1M CaCl2 is as good as P1 with RNase for removing RNA.

• 10% PEG is good to precipitate plasmid DNA. Isopropanol is not good.

Precipitation of RNA using Ammonium Acetate

1. Spin down 500 ml cells

2. Add 20 ml of P1 (50 mM Tris-HCl, 10 mM EDTA pH8.0) without RNase.

3. Add 20 ml of P2 (200 mm NaOH, 1% SDS solution), lyse for about 5 min.

4. Add 20 ml of P3 (7M ammonium acetate).

5. Incubation at 4 degrees overnight

The high salt precipitation may involve adding 1M potassium acetate and 7M ammonium acetate (concentrations of both may be varied, with higher concentrations of ammonium acetate being better for RNA precipitation) and holding the temperature at 4 degrees Celsius for approximately 6 to 12 hours to precipitate RNA (times and temperatures may be varied; lower temperatures and longer times improve precipitation and incubation at 4 degrees Celsius provides a higher percentage of precipitation). The ability to precipitate RNA during neutralization of the alkaline lysate helps eliminate the need for RNase. The acetate solution does not need to be pH adjusted and the pH is approximately 8. Conventional techniques using 3M potassium acetate at pH 5 are dramatically less effective at RNA precipitation and provide greater risk of DNA damage.

The phenyl 650-S resin (100 mL) was packed into a 5 cm diameter column to a height of 5 cm. The column was equilibrated with approximately 500 mL of 1.75M (NH₄)₂SO₄ at 20 mL/minute. Then 350 mL of the DEAE elution pool containing plasmid DNA was diluted with an equal volume of 3.5M (NH₄)₂SO₄ which results in a final concentration of 1.75M (NH₄)₂SO₄. The entire 700 mL of plasmid DNA in 1.75M (NH₄)₂SO₄ was pumped through the HIC column and the flow through peak which contains the plasmid was saved for further processing by ultrafiltration and diafiltration (UF/DF). The bound RNA, chromosomal DNA, denatured plasmid and endotoxins were eluted in a Milli-Q water wash followed by a 70% ethanol (v/v) wash.

Plasmid should not bind, genomic DNA, RNA should bind to phenyl 650-S.

Testing Results

9/5/2012 Test Tosoh Butyl 650M and Pall MEP resins

Use 2 ml culture and spin column. resin: 100 ul

1. Hangzhou miniprep spin column, eluted with 100 ul H₂O

2. Tosoh Butyl 650M eluted with 100 ul H₂O

3: Pall MEP eluted with 100 ul H₂O

4. Pall MEP eluted with 100 ul 2.5 M NaCl

	1	2	3	4
	Hangzhou Plasmid Spin Column	Butyl 650M	PALL MEP eluted with water	PALL MEP eluted with 2.5 M NaCl
OD260	18.6 ng/ul	60.6 ng/ul	12 ng/ul	39.8 ng/ul
260/280	1.27	1.58	1.81	2.08

http://www.freepatentsonline.com/6953686.pdf