Editing Plasmid MaxiPrep Q Membrane (section)

== 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]]