產品介紹

The E.Z.N.A.? Bacterial DNA Kit allows the rapid and reliable isolation of high-quality total cellular DNA from a wide variety of gram-positive and negative bacterial species. This kit uses an optimized lysis condition and up to 1 x 10^9 bacterial cells can be processed for each column. There are no organic extractions, thus reducing plastic waste and hands-on time to allow multiple samples to be processed in parallel. Bacterial cells are grown to log-phase and harvested. The cell wall is removed by lysozyme digestion and bead beating, followed by protease digestion. Following lysis, binding conditions are adjusted and the sample is applied to a HiBind DNA spin-column. Two rapid wash steps remove trace salts and protein contaminants, and DNA is finally eluted in water or Elution Buffer. Purified DNA can be directly used in downstream applications without the need for further purification.

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流程

DNA Extraction and Purification from up to 3 mL LB Culture

This method allows genomic bacterial isolation from up to 3 mL LB culture.

1. Culture bacteria in LB media to log-phase. (Overnight culture can be used in many cases).

2. Centrifuge no more than 3 mL culture or 1 x 10? cells at 4,000g for 10 minutes at room temperature. Aspirate and discard the media.

3. Add 100 μL TE Buffer. Vortex to completely resuspend the pellet.

4. Add 10 μL Lysozyme resuspended with Elution Buffer (see D3350 product manual for instructions). Incubate at 37°C for 10 minutes. The amount of enzyme required and/or the length of incubation may need to be modified depending on the bacterial strain used. Complete digestion of the cell wall is essential for efficient lysis. Longer incubation time may yield better results.

      Optional: Follow the short protocol below for difficult to lyse bacteria.
        1. Add 25 mg Glass Beads S to a 1.5 mL microcentrifuge tube (not provided).
        2. Add sample to the Glass Beads S.
        3. Vortex at maximum speed for 5 minutes.
        4. Let sample stand to allow the beads to settle.
        5. Transfer supernatant to a new 1.5 mL microcentrifuge tube.

5. Add 100 μL TL Buffer and 20 μL Proteinase K Solution. Vortex to mix thoroughly. Incubate at 55°C in a shaking water bath. Usually no more than 1 hour is required for bacterial lysis. If a shaking water bath is not available, incubate the samples and shake or briefly vortex every 20-30 minutes.

6. Add 5 μL RNase A. Invert tube several times to mix. Let sit at room temperature for 5 minutes.

7. Centrifuge at 10,000g for 2 minutes to pellet any undigested material. Transfer the supernatant to a new 1.5 mL microcentrifuge tube. Do not disturb the pellet.

8. Add 220 μL BL Buffer. Vortex to mix thoroughly. Incubate at 65°C for 10 minutes. A wispy precipitate may form upon addition of BL Buffer; it does not interfere with DNA recovery.

9. Add 220 μL 100% ethanol. Vortex for 20 seconds at maximum speed to mix thoroughly. Break any precipitates by pipetting up and down 10 times.

10. Insert a HiBind? DNA Mini Column into a 2 mL Collection Tube. Transfer the entire sample to the HiBind? DNA Mini Column, including any precipitate that may have formed.

11. Centrifuge at 10,000g for 1 minute. Discard the filtrate and the collection tube.

12. Insert the HiBind? DNA Mini Column into a new 2 mL Collection Tube.

13. Add 500 μL HBC Buffer diluted with 100% isopropanol (see the bottle for instructions). Centrifuge at 10,000g for 1 minute. Discard the filtrate and reuse the collection tube.

14. Add 700 μL DNA Wash Buffer diluted with 100% ethanol (see the bottle for instructions). Centrifuge at 10,000g for 1 minute. Discard the filtrate and reuse the collection tube.

15. Repeat Step 14 for a second DNA Wash Buffer step.

16. Centrifuge the empty HiBind? DNA Mini Column at maximum speed (≥10,000g) for 2 minutes to dry the column. This step is critical for removal of trace ethanol that may interfere with downstream applications.

17. Insert the HiBind? DNA Mini Column into a new nuclease-free 1.5 mL microcentrifuge tube.

18. Add 50-100 μL Elution Buffer heated to 65°C. Let sit for 3-5 minutes at room temperature. Yields may be increased by incubating the column at 65°C. Centrifuge at 10,000g for 1 minute to elute the DNA.

19. Repeat Step 18 for a second elution step.

20. Store eluted DNA at -20°C.