PlasmidFastTM Plasmid Purification Kit - 881

Directions for Use and Application Notes

• Reagent 1 - Cell Resuspension Buffer

• Reagent 2 - Cell Lysis Buffer

• Reagent 3 - Protein Removal Buffer

• Reagent 4 - RNase A Solution (IMPORTANT - For best results, store this material at -20 C upon arrival)

• Reagent 5 - Plasmid Precipitation Reagent

• Reagent 6 - Sterile TE buffer

• Overnight bacterial culture containing desired plasmid

• Clean, sterile microcentrifuge tubes (2 per prep)

• 37 °C water bath

• Ice bucket

• Microcentrifuge, capable of 10,000 -12,000 X g

• 70% Ethanol, Stored at room temp.

Important: Read all instructions and application notes before beginning this procedure.

1. Inoculate a bacterial culture, containing the desired plasmid, into 5 ml or more of sterile growth medium. Add appropriate antibiotics and grow overnight (at least 16 hours for maximum yield) at 37° C with vigorous shaking.

2. Cool overnight culture to 4 °C and transfer 1 - 1.5 ml of culture into a new, clean microcentrifuge tube. Centrifuge 30 seconds to pellet cells. Remove supernatant by decanting. Centrifuge an additional 5 seconds, and remove residual media by pipetting.

3. Add 200 L Reagent 1 (Cell Resuspension Buffer) and resuspend cells by gently pipetting up and down. (Note: Vortexing at this stage often results in poor cell resuspension and is not a recommended method.)

4. Add 200 L Reagent 2 (Cell Lysis Buffer) and immediately vortex to mix. Allow tube to stand at room temperature for 2 minutes to effect cell lysis.

5. Add 300 L of Reagent 3 (Protein Removal Buffer) and immediately vortex to mix. Incubate on ice for 5 minutes.

6. Centrifuge 5 minutes at room temperature and carefully transfer the supernatant to a fresh tube.

7. Add 5 L of Reagent 4 (RNase A solution) and incubate for 5 minutes at 37 °C.

8. Add 500 L of Reagent 5 (Plasmid Precipitation Reagent) and vortex to mix.

9. Centrifuge 5 minutes at room temperature. Remove as much of the supernatant as possible by pipetting. A brief (10 second) additional centrifugation may facilitate removal of the supernatant.

10. Add 400 L of 70% Ethanol, vortex briefly, and centrifuge for 2 minutes.

11. Decant the supernatant and dry pellet under vacuum for 5 minutes, or on the bench top for 30 minutes or until no odor of ethanol remains.

12. Resuspend the pellet in 25 L of sterile TE buffer.

Step 1: As when using any plasmid purification method, appropriate selection of bacterial strain, media, and antibiotic can drastically affect the yield of plasmid. Additionally, insufficient aeration of the overnight culture can severely curtail bacterial growth and yield of plasmid.

Chloramphenicol may be used to increase plasmid production in cultures, if desired.

Step 2: Complete removal of bacterial media at this step will greatly decrease risk of protein contamination in the final plasmid suspension.

Step 3: Complete resuspension of cells at this stage is critical to obtaining good yields. Pipetting the cells to mix ensures that there are no "pockets" of cells that are later not exposed to the lysis reagent.

Step 4: The cell suspension should appear clear and thick after this incubation, due to the release of bacterial genomic DNA into the solution.

Step 5: Addition of this reagent results in immediate formation of a thick, white flocculant. For best results, the tube should be mixed immediately after adding Reagent 3. The ice incubation is important for complete removal of contaminating bacterial genomic DNA from the plasmid prep.

Step 6: Some of the precipitate from this centrifugation may remain on top of the supernatant. Use care in transferring the supernatant to a fresh tube. If substantial amounts of the white insoluble matter are carried over with the supernatant, centrifuge briefly to pellet this material and transfer the supernatant to a fresh microcentrifuge tube.

Step 7: This step is optional, and can be omitted if RNA contamination in the plasmid is not problematic.

Step 8: Thorough mixing at this stage is important for obtaining optimum yields.

Step 9: Centrifuge speed can drastically affect precipitation of plasmid. Too high centrifuge speed (> 12,000 X g) can result in significant co-precipitation of polysaccharide material that can later inhibit some enzyme digestions. Speeds too low can result in low yields. If your centrifuge cannot accommodate 10,000 - 12,000 X g, plasmid may be best precipitated by alcohol precipitation (See Below).

Step 10: The pellet at this stage should be fairly small and very slightly off-white. A very large, white pellet may indicate that there was co-precipitation of polysaccharide with the DNA. If desired, the pellet may be resuspended in the ethanol by pipetting before centrifugation. This is especially important if the pellet is significantly larger than expected. Resuspending the pellet at this stage will help remove any contaminating polysaccharide.

Step 11: Do not allow the pellet to dry for too long; the pellet may be difficult to resuspend.

Step 12: If the pellet in Step 10 was much larger than expected, centrifuge the resuspended material briefly and transfer the supernatant to a fresh tube. The pelleted material will contain any residual polysaccharide that may have co-precipitated.

Optional Alcohol Precipitation:

After incubation with RNase A in Step 7, add 1.2 volumes of room temperature Isopropanol or ice-cold ethanol. Vortex to mix and immediately centrifuge at 4 °C for 10 minutes (centrifuge speed in excess of 8,000 X g).

Decant alcohol and rinse pellet once in 200 L 70% ethanol. Centrifuge an additional 2 minutes.

Remove alcohol and dry pellet inverted for 30 minutes (5 minutes under vacuum) or until no strong odor of ethanol remains.

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