Valencia/Mini prep

From 2007.igem.org

Latest revision as of 06:40, 26 September 2007

Information obtained from:

Roche Applied Science: High Pure Plasmid Isolation Kit

Protocol obtained from: High Pure Plasmid Isolation Kit

Pdf link: Pdf

Protocol for preparing DNA from 0.5 - 4.0 ml of E. coli culture with a density of 1.5-5.0 A600 units per ml.

Scaling up to 10 ml is possible, nothing has to be modified in the protocol, the volumes of the solutions stay the same, as higher volumes would affect the capacity of the columns. The yield depends on the growing conditions of the strain and the lysis efficiency as seen in the table of experimental results.

You must place the Binding Buffer on ice before starting the procedure.

1. Place Binding Buffer on ice.
2. Prepare the starting material:
   • Pellet the bacterial cells from 0.5 - 4.0 ml of E. coli culture.
     • The cells should have a density of 1.5 - 5.0 A600 units per ml.
   • Discard the supernatant.
   • Add 250 μl Suspension Buffer + RNase to the centrifuge tube containing the bacterial pellet.
   • Resuspend the bacterial pellet and mix well.
3. Treat the resuspended bacterial pellet as follows:
   • Add 250 μl Lysis Buffer.
   • Mix gently by inverting the tube 3 to 6 times.
     • To avoid shearing genomic DNA, do not vortex!
   • Incubate for 5 min at any temperature between +15°C and +25°C.
     • Do not incubate for more than 5 min!
4. Treat the lysed solution as follows:
   • Add 350 μl chilled Binding Buffer.
   • Mix gently by inverting the tube 3 to 6 times.
   • Incubate on ice for 5 min.
     • The solution should become cloudy and a flocculant precipitate should form.
     • Centrifuge for 10 min at approx. 13,000 x g (full speed) in a standard tabletop microcentrifuge
5. After centrifugation:
   • Insert one High Pure Filter Tube into one Collection Tube.
   • Transfer entire supernatant from Step 5 into upper buffer reservoir of the Filter Tube.
   • Insert the entire High Pure Tube assembly into a standard tabletop microcentrifuge.
   • Centrifuge for 1 min at full speed.
6. After centrifugation:
   • Remove the Filter Tube from the Collection Tube, discard the flowthrough liquid, and re-insert the Filter Tube in the same Collection Tube.
   • If the E. coli strain in Step 2 has a high nuclease content (e.g.,HB101 or JM strains), perform the optional wash step below before going to Step 7.
   • If the E. coli strain in Step 2 does not have a high nuclease content (e.g., XL1 blue or DH5 strains), skip the optional wash step and perform Step 7.
     • Optional wash step: To eliminate high nuclease activity from the preparation:
     • Add 500 μl of Wash Buffer I to the upper reservoir of the Filter Tube.
     • Centrifuge for 1 min at full speed and discard the flowthrough.
7. To wash the cells:
   • Add 700 μl Wash Buffer II to the upper reservoir of the Filter Tube.
   • Centrifuge for 30 - 60 s at full speed and discard the flowthrough.
8. After discarding the flowthrough liquid:
   • Centrifuge the entire High Pure tube assembly for additional 1 min.
   • Discard the Collection Tube.
     • The extra centrifugation time ensures removal of residual Wash Buffer.
9. To elute the DNA:
   • Insert the Filter Tube into a clean, sterile 1.5 ml microcentrifuge tube.
   • Add 100 μl Elution Buffer or double dist. water (pH adjusted to 8.0 -8.5) to the upper reservoir of the Filter Tube.
   • Centrifuge the tube assembly for 1 min at full speed.
10. The microcentrifuge tube now contains the eluted plasmid DNA.
    • Either use the eluted DNA directly in such applications as cloning or sequencing or store the eluted DNA at +2 to +8°C or -15 to -25°C for later analysis.