Editing Plasmid MaxiPrep Q Membrane (section)

== 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]] 

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