Paul.xu at 21:47, 4 March 2013

2013-03-04T21:47:56Z

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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 (with RNase A). Usually 15 ml P1 is good enough. Make sure all cells are suspended, no visible aggregate---This is very critical. You can use a clean spoon to disrupt the pellet.

4. Lyse cells with 10 ml to 20 ml of P2/per gram of pellet. Usually 15 ml P2 is good enough. Swirl the centrifuge tubes gently until cells are completely lysed. Do not shake.

Incunbate at RT for 3 min.

5. Neutralize with cold 10 ml to 20 ml of P3/per gram of pellet. Usually 15 ml P3 is good enough.

Make sure you mix the solution gently in the begining and then more vigorously, until all gooey lysate becomes loose fluffy white material (lysate becomes not viscous at all).

6. Add 1 ml P1, incubate at 37 oC for 1 hour.

7. Incubate on ice for 15 to 30 min. 

8. Spin at 6000 rpm for 15 min using 400 ml centrifuge tube and Superlit rotor.

If the pellet is not lose,  transfer supernatant to 50 ml tubes, Spin at 12000 rpm for 10 min, high speed centrifuge, collect supernatant, filter liquid using a coffee filter.

9. Add 1/20 volume (1.5 ml for 30 ml supernatant) of 20% Triton X-114 (in 80% ethanol), incubate on ice for 30 minutes, mix occasionally.

1.2% Triton X-114+ 0.6% [w/v] SDS===> not cloudy at RT

Other detergents: Nonidet-P40 (Igepal CA 630), Triton X-100, Tween 20

10. Clean used Qiagen Maxiprep column with 30 ml CIP solution: 1.5M NaCl, 0.1% NaOH. Wash with 30 ml water.  Equilibrate the column with 15 ml of QBT buffer.

11. Load supernatant to the column. Do not discard flow through.

12. Wash with 2 X of 30 ml QC buffer.

13. Elute with 15 ml QF or QN buffer. 14. Clean column with 30 ml of CIP solution. Wash out NaCl with 30 ml of water.

15. Equilibrate Maxiprep column with 15 ml QBT.

16. Repeat  steps 11 to 14, discard flow through at step 11.

17. Wash column with  10 ml 20% EtOH. Store the column in 20% EtOH at 4 oC.

 18. Measure DNA concentration using nanodrop.

19. Precipitate DNA using 0.7 vol (11 ml) of isopropanol, incubation on ice for 15 to 30 min.

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

Inside a biosafety hood:

21. Decant solution, add 1ml of  70% ethanol (by spray ~2 to 4 ml of 70% ethanol). Decant enthanol gently. Be careful not to disturb pellet.

22. Transfer pellet in 70% ethanol to a clean 1.5 ml tube. Spin brieftly, remove ethanol.  Dissolve DNA in 500 ul TE

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

 

[[Image:Maxiprep_with_used_qiagen_column.JPG]] 

{| border="1" cellspacing="1" cellpadding="1" width="200"
|-
| Lane 
| Clone ID 
| Name 
| ng/ul 
| OD260/280 
| Total ug 
|-
| 1 
| 537 
| psPAX2-D64V 
| 1330 
| 1.91 
| 665 
|-
| 2 
| 469 
| pAAV.CMV-Cre-RFP 
| 1306 
| 1.91 
| 653 
|-
| 3 
| 533 
| pLVX-Cre-IRES-EGFP 
| 1479 
| 1.93 
| 739 
|-
| 4 
| 249 
| pAAV-DJ 
| 1287 
| 1.93 
| 643 
|-
| 5 
| 248 
| pAAV-Helper 
| 1663 
| 1.95 
| 831 
|-
| 6 
| 527 
| pLVX-EF1-Cre-IRES-EGFP 
| 1526 
| 1.93 
| 763 
|-
| 7 
| 527 
| pLVX-EF1-Cre-IRES-EGFP 
| 1710 
| 1.93 
| 855 
|-
| 8 
| 533 
| pLVX-Cre-IRES-EGFP 
| 1309 
| 1.95 
| 654 
|-
| 9 
| 461 
| pAAV-CMV-Cre-mKate2 
| 323 
| 1.91 
| 80 
|}

 

[[Category:Protocols]]

Plasmid MaxiPrep Used Qiagen Maxiprep Column - Revision history

Paul.xu at 21:47, 4 March 2013