Paul.xu at 23:58, 12 November 2012

2012-11-12T23:58:37Z

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==   Gel Filtration + Q membrane ==

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 cold 10 ml to 20 ml P1 buffer/per gram of pellet, if less than 2 g, use 10 ml of P1.  Use a stainless steel spatula and votex to break big pellet into homogeneous supension. 

4. Lyse cells with  10 ml to 20 ml of P2/per gram of pellet. Mix gently until cells are completely lysed. Incubate at RT for 5 min to digest RNA. 

5. Neutralize with cold 10 ml to 20 ml of P3/per gram of pellet. Mix gently in the begining, then votex a few seconds. 

6. If P3 without acetic acid or ammonium acetate is used, you can incubate the suspension 4 hours to o/n to precipitate RNA.

7. Spin for 5 min at 6000 rpm, Transfer supernatant to 50 ml tubes, Spin at 12000 rpm for 10 min using high speed centrifuge, collect supernatant, filter liquid using a coffee filter paper. 

8. Add 0.7 volume of isopropanol, incubate on ice for 5~15 min. Do not inubate for too long as protein and other junk will co-precipitate with DNA/RNA. Spin at 12000 rpm for 15 min.

9. Wash pellet by spraying with 1 ~3 ml 70% ethanol. Drain liquid each time. Put tube on top of paper towel for a few minutes.  Resuspend pellet in 4 ml (Total <4.5 ml) Buffer A or TE. Pellet may not be soluble, so use 1 ml filter pipette tip to disrupt the pellet into smaller pieces.

10. Add 10~15 ul of 50 mg/ml RNAse A, incubate at 37 oC for 1 hour or RT for o/n. The insoluble RNA/protein/DNA pellet should have been dissolved. Hopefully most of the RNA should be degraded. 

11. Transfer  ~1.3 ml to 1.5 ml microcentrifuge tubes. Label them! Be careful not to cross contaminate or knock-over. 

12. Add ~75 ul of 10% Triton X-114 to each tube (do not need to be accurate, do not need to change tip, do not corss contaminate or knock-over, just add to the top of tubes, do not touch the inside of tubes), close the lids, mix for a few seconds by votexing, incubate on ice for 3 min. 

13. Heat at 55 oC for 5 min. 

14. Spin at 13000 rpm for 3 min. 

15. Transfer supernatant to a clean tube. DO NOT take white interface (protein) and bottom phase. 

16. Load <=5 ml to a BioRad (Bio-Sil TSK250) (Tosoh) HPLC gel filtration column, separate RNA from DNA using program 47,  filtered Buffer D (10 mM Tris-HCl, pH7.5, 1 mM EDTA, 50 mM NaCl). Flow rate: 2.5 ml/min, 4 ml per fraction. DNA should come out at fraction 21. 

 

Be very careful to avoid cross contamination, before injection of plasmid solution:

*Wash the enternal injection valve extensively with clean ddH2O
*Wash injection syringe at least 5 times.
*Wash injection loop at least 5 times

 

17. Combine the first peak fractions containing plasmid DNA.

Wash glass tubes immediately with ddH2O at least 4 times. Dry them top-down to avoid dust.

After FPLC, wash injection valve, loop, syringe and needle 5 times with water.

 

18. Activate Q membrane with 4 ml of 1 M NaOH then 10 ml of 2 M NaCl.

19. Equilibrate Q membrane with '''Buffer A''' (0.5 M NaCl in 10 mM Tris buffer, 1 mM EDTA, pH 8.0.) 

'''SartoBind Q:''' Dissolve DNA in & washing with '''0.5 M''' NaCl in 10 mM Tris buffer, 1 mM EDTA, pH 8.0. 

20. Load  DNA solution to one equilibrated <strike>Mustung</strike> '''SartoBind''' Q membrane disk using a syringe of peristaltic pump at  flow rate of 1 ~ 4 ml/min. 

21. Wash disk with '''5''' ml of '''Buffer B '''(25 mM Tris-HCl, '''0.5''' 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. 

This wash will elute endotoxin and residual RNA. 

22. Wash with 20 ml of '''Buffer A'''  (0.5 M NaCl, 10 mM Tris buffer and 1 mM EDTA at pH 8). 

This wash is to wash out Triton X-114 and residual RNA 

23. Elute plasmid DNA slowly using 15~ 20 ml of '''Buffer C''' ('''1.3 M''' NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA), Discard the first 1 ml, Soak Q membrane in this buffer for 3 min, then slowly (0.5 ml/min) elute DNA using peristaltic pump. Collect elute in 50 ml tube. 

'''SartoBind Q:''' Clean Q membrane with 5 ml of 1 M NaOH, then  10 ml of 2.5 M NaCl.  Wash Q membrane with 10 ml water. Do not discard the membrane. It is about $250 each. 

 

24. Measure DNA concentration using nanodrop. 

25. Precipitate DNA using 0.7 vol (14 ml) of isopropanol,  incubation on ice for 15 to 30 min. 

26. Spin down DNA using high speed centrifuge 12000 rpm for 15 min. 

'''Inside a biosafety hood:''' 

27. Wash 2X with 2 ml 70% ethanol (by spray ~2 to 4 ml of 70% ethanol). Decant enthanol. 

28. Dissolve DNA in 5 X 200 ul TE 

29. Measure DNA concentration and OD260/280 ratio, check DNA quality by agarose gel electrophoresis. 

== CaCl2 + PEG + Q membrane ==

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 10 ml to 20 ml P1 buffer/per gram of pellet, if less than 2 g, use 10 ml of P1. 

4. Lyse cells with 10 ml to 20 ml of P2/per gram of pellet, '''incubate at RT for < 5 min.''' This will digest RNA 

5. Neutralize with 10 ml to 20 ml of P3/per gram of pellet. 

6. Add 5 M CaCl2 '''slowly while mixing''' to  adjust  final concentration of 0.5 M (up to 1.4M). Incubate at RT for 15 min. 

 

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

If you want to skip step 8 & 9, you must dilute the filtrate with 3 volume of water. Otherwise DNA will not bind. 

 

8. Add 0.7 volume of isopropanol, incubate on ice for 15 min. Spin at 12000 rpm for 15 min. 

9. Wash pellet with 1 ~3 ml 70% ethanol. Dissolve pellet in 4 X0.5 ml (Total 2 ml) Buffer A.

'''Optional:''' Precipitate DNA by adding 40% PEG to final concentration of 10%. Spin at 12000 rpm for 10 min to precipitate plasmid DNA, discard supernatant and wash DNA with 70% ethanol. Dissolve DNA in TE. 

10. Activate membrane with 4 ml of 1 M NaOH then 10 ml of 2 M NaCl.

11. Equilibrate membrane with '''Buffer A''' (0.6 M NaCl in 10 mM Tris buffer, 1 mM EDTA, pH 8.0.) 

'''SartoBind Q:''' Dissolve DNA in & washing with '''0.6 M''' NaCl in 10 mM Tris buffer, 1 mM EDTA, pH 8.0. 

12. Load  DNA solution to one equilibrated <strike>Mustung</strike> '''SartoBind''' Q membrane disk using a syringe of peristaltic pump at  flow rate of 1 ~ 4 ml/min. 

13. Wash disk with '''5''' ml of '''Buffer B '''(25 mM Tris-HCl, '''0.6''' 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. 

This wash will elute endotoxin and RNA. 

14. Wash with 20 ml of '''Buffer A'''  (0.6 M NaCl, 10 mM Tris buffer and 1 mM EDTA at pH 8). 

This wash is to wash out Triton X-114 and residual RNA 

15. Elute plasmid DNA slowly using 15 ml of '''Buffer C''' (1.3 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA), Discard the first 1 ml, Soak membrane in this buffer for 3 min, then slowly (0.5 ml/min) elute DNA using peristaltic pump. Collect elute in 50 ml tube. 

'''SartoBind Q:''' Clean membrane with additional 10 ml of 2.5 M NaCl after eluting with buffer C containing 1.3 M NaCl. 

Regenerate disk using 5 ml of 1 M NaOH and then 5 ml of 2 M NaCl. Wash membrane with 10 ml water. Do not discard the membrane. It is about $250 each. 

 

16. Measure DNA concentration using nanodrop. 

17. Precipitate DNA using 0.7 vol (15 ml) of isopropanol,  incubation on ice for 15 to 30 min. 

18. Spin down DNA using high speed centrifuge 12000 rpm for 15 min. 

'''Inside a biosafety hood:''' 

19. Wash 2X with 70% ethanol (by spray ~2 to 4 ml of 70% ethanol). 

20. Dissolve DNA in 5 X 200 ul TE 

21. Measure DNA concentration and OD260/280 ratio, check DNA quality by agarose gel electrophoresis. 

 

 

== Protocol 1: 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 10 ml to 20 ml P1 buffer/per gram of pellet, if less than 2 g, use 10 ml of P1. 

4. Lyse cells with 10 ml to 20 ml of P2/per gram of pellet, '''incubate at RT for < 5 min'''. This will digest RNA 

5. Neutralize with 10 ml to 20 ml of P3/per gram of pellet. 

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

7. Activate membrane with 4 ml of 1 M NaOH then 10 ml of 2 M NaCl.

8. Equilibrate membrane with '''Buffer A''' (0.6 M NaCl in 10 mM Tris buffer, 1 mM EDTA, pH 8.0.) 

'''SartoBind Q:''' Washing with '''0.6 M''' NaCl in 10 mM Tris buffer, 1 mM EDTA, pH 8.0. 

9.'''Dilute DNA solution with 3 volume of water.''' Load  diluted DNA solution to one equilibrated <strike>Mustung</strike> '''SartoBind''' Q membrane disk using a syringe of peristaltic pump at  flow rate of 1 ~ 4 ml/min. 

10. Wash disk with '''5''' ml of '''Buffer B '''(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. 

This wash will elute endotoxin and RNA. 

11. Wash with 20 ml of '''Buffer A'''  (0.6 M NaCl, 10 mM Tris buffer and 1 mM EDTA at pH 8). 

This wash is to wash out Triton X-114 and residual RNA 

12. Elute plasmid DNA slowly using 20 ml of '''Buffer C''' (1.3 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA), Discard the first 1 ml, Soak membrane in this buffer for 3 min, then slowly (0.5 ml/min) elute DNA using peristaltic pump. Collect elute in 50 ml tube. 

'''SartoBind Q:''' Clean membrane with additional 10 ml of 2.5 M NaCl after eluting with buffer C containing 1.3 M NaCl. 

Regenerate disk using 5 ml of 1 M NaOH and then 5 ml of 2 M NaCl. Wash membrane with 10 ml water. Do not discard the membrane. It is about $250 each. 

 

13. Measure DNA concentration using nanodrop. 

14. Precipitate DNA using 0.7 vol (15 ml) of isopropanol,  incubation on ice for 15 to 30 min. 

15. Spin down DNA using high speed centrifuge 12000 rpm for 15 min. 

'''Inside a biosafety hood:''' 

16. Wash 2X with 70% ethanol (by spray ~2 to 4 ml of 70% ethanol). 

17. Dissolve DNA in 5 X 200 ul TE 

18. Measure DNA concentration and OD260/280 ratio, check DNA quality by agarose gel electrophoresis. 

 

From 1 L culture you should get a least 1 mg DNA from SartoBind Q 75 membrane. 

=== Method Validation ===

Date: 11/4/2012 by Paul 

Clone #1385, from 250 ml o/n culture. Use 10 ml of P1, P2, P3 each. 

Load 5 ul from each fraction to 1% gel.

[[Image:Plasmid Sartobind Q membrane 11 4 2012.JPG]] 

{| width="378" cellspacing="1" cellpadding="1" border="1"
|-
| Fraction 
| 1 
| 2 
| 3 
| 4 
| 5 
| 6 
| 7 
| 8 
| 9 
| 10 
| 11 
| 12 
| 13 
| 14 
|-
| OD260 ng/ul 
| 35 
| 167 
| 134 
| 149 
| 171 
| 147 
| 146 
| 157 
| 140 
| 132 
| 100 
| 92 
| 110 
| 80.9 
|-
| 260/280 
| 1.86 
| 1.95 
| 1.97 
| 1.99 
| 1.98 
| 1.98 
| 1.93 
| 1.99 
| 2.01 
| 1.99 
| 1.99 
| 1.99 
| 1.98 
| 2.0 
|-
| Fraction 
| 15 
| 16 
| 17 
| 18 
| 19 
| 20 
| 21 
| 
| 
| 
| 
| 
| 
| 
|-
| OD260 ng/ul 
| 334 
| 257 
| 208 
| 139 
| 109 
| 83 
| 62 
| 
| 
| 
| 
| 
| 
| 
|-
| 260/280 
| 
| 1.93 
| 
| 1.95 
| 1.95 
| 1.95 
| 1.97 
| 
| 
| 
| 
| 
| 
| 
|}

Final: 

Toal: 1.2 ml, concentration:.2273.5 ng/ul, 260/280=1.92 

 

11/6/2012: By Yingying. From 1 L culture

Load liquid after precipitation and filtration.

Wash with 5 ml Buffer A( 0.75M NaCl + 0.5% Triton X-114), then 15 ml Buffer B (0.6 M NaCl)

 

[[Image:Sartobind q plasmid 11 6 2012.jpg]]

 

== Protocol 2: 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 10 ml P1 buffer/per gram of pellet, if less than 2 g, use 10 ml of P1. 

4. Lyse cells with 10 ml of P2/per gram of pellet 

5. Neutralize with 10 ml of P3/per gram of pellet. 

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

7. Precipitate DNA/RNA using 0.7 volume of isopropanol, incubate on ice for 15 min. Spin at 12000 rpm for 15 min. Dissolve DNA in 5 X 1 ml of Buffer A, Spin at 13000 rpm microcentrifuge to precipitate insoluble matter. 

8. Activate membrane with 4 ml of 1 M NaOH then 4 ml of 1 M NaCl. 

9. Equilibrate membrane with '''Buffer A''' (0.5 M NaCl in 10 mM Tris buffer, 1 mM EDTA, pH 8.0.) 

'''SartoBind Q:''' Dissolve DNA in & washing with'''0.6 M''' NaCl in 10 mM Tris buffer, 1 mM EDTA, pH 8.0. 

10. Load  DNA solution to one equilibrated Mustung Q membrane disk using a syringe of peristaltic pump at  flow rate of 1 ~ 4 ml/min. 

11. Wash disk with 5 ml of '''Buffer B '''(25 mM Tris-HCl, 0.5 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. 

12. Wash with 10 to 15 ml of '''Buffer A'''  (0.5 M NaCl, 10 mM Tris buffer and 1 mM EDTA at pH 8). 

13. Elute plasmid DNA slowly using 5~10 ml of '''Buffer C''' (1.5 M NaCl, 10 mM Tris buffer, 1 mM EDTA, pH 8), collect fractions. 

'''SartoBind Q:''' Elute with 3M NaCl after eluting with 1.5 M NaCl. Check DNA quality vs 1.5 M NaCl elution. 

Regenerate disk using 4 ml of 1 M NaOH and then 4 ml of 2 M NaCl. 

14. Measure DNA concentration using nanodrop. 

15. Precipitate DNA using 0.7 vol of isopropanol and incubation on ice for 15 to 30 min. 

16. Spin down on microcentrifuge for 10 min. 

'''Inside a biosafety hood:''' 

17. Wash with 70% ethanol. 

18. Dissolve DNA in 3 X 200 ul TE 

19. Measure DNA concentration and OD260/280 ratio, check DNA quality by agarose gel electrophoresis. 

 

 Plasmid MaxiPrep Q Membrane using Mustang Q menbrance. 

 

=== By Xiuli 10/23/2012: 1382 Maxiprep with Mustung Q Membrane ===

 Sample : 1382, 260ml,  OD=2.04

Adding 10 ml P1 (with RNase) +10ml P2+10ml P3, following the protocol except washing buffer B (0.75 M NaCl).

Elute with buffer C (1.6M NaCl), collect 1ml fraction 1, 5 ml fraction 2 and 4 ml fraction 3. measure DNA using nanodrop.

Fraction 1= 3ug/ml: fraction 2= 48.7ug/ml, fraction 3=10 ug/ml

combine fraction 2 and 3 (280 ug DNA)  and precipitate with 0.7 Volume isopropanal.

Centrifuge at 15000 rpm for 25 mins.

Can see a  little white, glassy pellet, wash with 70% ethenal, dissove in 300 ul TE buffer. The conc. is 100ng/ul.  totally get 30ug DNA, DNA recovery is very low, about 10-15 % after precipitation.

need to be dissolve in less volume to get high Conc. for next step transfection.

=== By XiuLi 11-02-2011 Maxiprep with SartoBind Q 75 Membrane ===

Maxi prep 1386 (250ml Culture), using StatoBind Q membrance, Elute  with 1ml 1.5 M NaCl (1-10) and 3M E11-E15) 

{| width="325" cellspacing="1" cellpadding="1" border="1"
|-
| Fraction
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
|-
| OD260
| 15.1
| 107
| 225
| 420
| 344
| 260
| 262
| 209 
| 144 
| 105 
|-
| 260/280 ratio
| 
| 
| 
| 
| 
| 
| 
| 
| 
| 
|}

 

Elute with 3M NaCl (E11-E15) 

 

{| width="325" cellspacing="1" cellpadding="1" border="1"
|-
| Fraction
| 11
| 12
| 13
| 14
| 15
|-
| OD260
| 85.
| 57.6
| 35.1
| 28
| 19.6
|-
| 260/280 ratio
| 
| 
| 
| 
| 
|}

[[Category:Protocols]]

Plasmid MaxiPrep Q Membrane - Revision history

Paul.xu at 23:58, 12 November 2012