Editing Plasmid MaxiPrep Q Membrane

== &nbsp; 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, <br> 

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.&nbsp; Use a stainless steel spatula and votex to break big pellet into homogeneous supension.<br> 

4. Lyse cells with&nbsp; 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.<br> 

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.<br> 

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. <br> 

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.&nbsp; Resuspend pellet in 4 ml (Total &lt;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.<br> 

11. Transfer&nbsp; ~1.3 ml to 1.5 ml microcentrifuge tubes. Label them! Be careful not to cross contaminate or knock-over.<br> 

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.<br> 

13. Heat at 55 oC for 5 min.<br> 

14. Spin at 13000 rpm for 3 min.<br> 

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

16. Load &lt;=5 ml to a BioRad (Bio-Sil TSK250) (Tosoh) HPLC gel filtration column, separate RNA from DNA using program 47,&nbsp; 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. <br> 

<br> 

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

<br> 

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. 

<br> 

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.)<br> 

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

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

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). <br> 

This wash contains Triton X-114, which has high UV absorbance, so there is no need to measure OD260.<br> 

This wash will elute endotoxin and residual RNA.<br> 

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

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

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. <br> 

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

<br> 

24. Measure DNA concentration using nanodrop.<br> 

25. Precipitate DNA using 0.7 vol (14 ml) of isopropanol,&nbsp; incubation on ice for 15 to 30 min.<br> 

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

'''Inside a biosafety hood:'''<br> 

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

28. Dissolve DNA in 5 X 200 ul TE<br> 

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

== 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, <br> 

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.<br> 

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

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

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

<br> 

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

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

<br> 

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

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. <br> 

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.)<br> 

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

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

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). <br> 

This wash contains Triton X-114, which has high UV absorbance, so there is no need to measure OD260.<br> 

This wash will elute endotoxin and RNA.<br> 

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

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

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. <br> 

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

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.<br> 

<br> 

16. Measure DNA concentration using nanodrop.<br> 

17. Precipitate DNA using 0.7 vol (15 ml) of isopropanol,&nbsp; incubation on ice for 15 to 30 min.<br> 

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

'''Inside a biosafety hood:'''<br> 

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

20. Dissolve DNA in 5 X 200 ul TE<br> 

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

<br> 

<br> 

== Protocol 1: 1 L culture<br>  ==

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

2. Spin down cells, <br> 

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.<br> 

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

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

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

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.)<br> 

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

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

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). <br> 

This wash contains Triton X-114, which has high UV absorbance, so there is no need to measure OD260.<br> 

This wash will elute endotoxin and RNA.<br> 

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

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

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. <br> 

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

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.<br> 

<br> 

13. Measure DNA concentration using nanodrop.<br> 

14. Precipitate DNA using 0.7 vol (15 ml) of isopropanol,&nbsp; incubation on ice for 15 to 30 min.<br> 

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

'''Inside a biosafety hood:'''<br> 

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

17. Dissolve DNA in 5 X 200 ul TE<br> 

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

<br> 

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

=== Method Validation<br>  ===

Date: 11/4/2012 by Paul<br> 

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

Load 5 ul from each fraction to 1% gel. 

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

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

Final:<br> 

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

<br> 

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) 

<br> 

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

<br> 

== Protocol 2: 1 L culture<br>  ==

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

2. Spin down cells, <br> 

3. Resuspend cells with 10 ml P1 buffer/per gram of pellet, if less than 2 g, use 10 ml of P1.<br> 

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

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

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

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.<br> 

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

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

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

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

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.<br> 

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

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. <br> 

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

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

14. Measure DNA concentration using nanodrop.<br> 

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

16. Spin down on microcentrifuge for 10 min.<br> 

'''Inside a biosafety hood:'''<br> 

17. Wash with 70% ethanol.<br> 

18. Dissolve DNA in 3 X 200 ul TE<br> 

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

<br> 

<br> Plasmid MaxiPrep Q Membrane using Mustang Q menbrance. <br> 

<br> 

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

<br> Sample&nbsp;: 1382, 260ml,&nbsp; 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)&nbsp; and precipitate with 0.7 Volume isopropanal. 

Centrifuge at 15000 rpm for 25 mins. 

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

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

{| 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<br> 
| 144<br> 
| 105<br>
|-
| 260/280 ratio 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|}

<br> 

Elute with 3M NaCl (E11-E15)<br><br> 

<br> 

{| 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 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|}

[[Category:Protocols]]