Plasmid DNA purification
5    NucleoSpin® Plasmid/ Plasmid (NoLid) protocols
5.1   Isolation of high-copy plasmid DNA from E. colt
Before starting the preparation:
•   Check if Wash Buffer A4 was prepared according to section 3. 1    Cultivate and harvest bacterial cells
Use 1-5 mL of a saturated E.coti LB culture, pellet cells in a standard benchtop microcentrifuge for 30 s at 11,000 x g. Discard the supernatant and remove as much of the liquid as possible.
Note: For isolation of iow-copy plasmids refer to section 5.2.
11,000 x g, 30 s
2    Cell lysis
Add 250 uL Buffer A1. Resuspend the cell pellet completely by vortexing or pipetting up and down. Make sure no cell clumps remain before addition of Buffer A2!
Attention: Check Buffer A2 for precipitated SDS prior to use. If a white precipitate is visible, warm the buffer for several minutes at 30-40 °C until precipitate is dissolved completely. Mix thoroughly and cool buffer down to room temperature (18-25 °C).
Add 250 uL Buffer A2. Mix gently by inverting the tube 5-8 times. Do not vortex to avoid shearing of genomic DNA. Incubate at room temperature for up to 5 min or until lysate appears clear.
Add 300 uL Buffer A3. Mix thoroughly by inverting the tube 6-8 times until blue samples turn colorless completely! Do not vortex to avoid shearing of genomic DNA!
Make sure to neutralize completely to precipitate all protein and chromosomal DNA. LyseControl should turn completely colorless without any traces of blue.
+ 250 uL A1 Resuspend
+ 250 uL A2 Mix RT, 5 min
+ 300 ul_ A3 Mix
Clarification of lysate
Centrifuge for 5 min at 11,000 x gat room temperature. Repeat this step in case the supernatant is not clear!
Ö
11,000 x g, 5-10 min
Bind DNA
Place a NucleoSpin® Plasmid/Plasmid (NoLid) Column in a Collection Tube (2 mL) and decant the supernatant tram step 3 or pipette a maximum ol 700 uL ot the supernatant onto the column. Centrifuge for 1 min at 11,000 x g. Discard flowthrough and place the NucleoSpin® Plasmid/Plasmid (NoLid) Column back into the collection tube.
Repeat this step to load the remaining lysate.
Load supernatant
11,000 x g, 1 min
Wash silica membrane
Add 600 uL Buffer A4 (supplemented with ethanol, see section 3). Centrifuge for 1 min at 11,000 x g. Discard flowthrough and place the NucleoSpin® Plasmid/Plasmid (NoLid) Column back into the empty collection tube.
+ 600 uL A4
11,000 x g, 1 min
6   Dry silica membrane
Centrifuge for 2 min at 11rQ00xg and discard the collection tube.
Note: Residual ethanolic wash buffer might inhibit enzymatic reactions.
11,000 x g, 2 min
Elute DNA
Place the NucleoSpin® Plasmid/Plasmid (NoLid) Column in a 1.5 mL microcentrifuge tube (not provided) and add 50 uL Buffer AE. Incubate for 1 min at room temperature. Centrifuge for 1 min at 11,000 x g.
Note: For more efficient eiution procedures and alternative eiution buffer (e.g., TE buffer or water) see section 2.5.
+ 50 uL AE RT, 1 min
11,000 x g, 1 min