Bacterial Genomic DNA Extraction Protocol

(From Omega Company)

1.This method allows genomic bacterial isolation from up to 3 ml LB culture. Grow Bacteria in LB media to log phase. (Overnight culture can be used in many cases.

2.Harvest no more than 3 ml culture by centrifugation at 4000 x g for 10 min at room temperature.

3.Remove medium completely and resuspend cells in 180 μl TE buffer. Add 20 μl of 50 mg/ml lysozyme solution, Incubate at 30℃ for 10 min.

Note: The amount of enzyme required and/or the incubation time may need to be modified depending on the bacterial strain used. Complete digestion of te cell wall is essential for efficient lysis. Longer incubation time might yield better results.

4.Pellet digested cell by centrifugation at 5000 x g for 5 min at room temperature. Discard the supertnatant and add 200 μl Buffer BTL. Vortex to resuspend cells.

Optional: For complete digestion of bacterial cell wall especially of Gram-Positive bacteria, add 25-40 mg Glass Powder and vortex at maximal speed for 5 minutes. Let it stand to allow the beads to settle. Transfer supernatant to a new 1.5 ml centrifuge tube (not supplied).

5.Add 25 μl Proteinase K solution and vortex to mix well. Incubate at 55℃ in a shaking water bath to effect complete lysis. Usually no more than 1 h is required for bacterial lysis. If no shaking waterbath is available, inbubate and shake or briefly vortex the samples every 20-30 minutes.

6.Add 5 μl RNase A to samples and invert tube weveral times to mix. Incubate at room temperature for 10-30 minutes.

(Optional) Centrifuge at 10000 x g for 5 min to pellet insoluble debris. Carefully aspirate the supernatant and transfer to a sterile microcentrifuge tube leabing behind any insoluble pellet.

7.Add 220 μl Buffer BDL and shake or briefly vortex to mix. Incubate at 65℃ for 10 minutes. A wispy precipitate may form upon addition of Buffer BDL; it does not interfere with DNA recovery.

8.Add 220 μl absolute ethanol (room temperature, 96-100%) and mix thoroughly by vortexing at maxi speed for 20 seconds. If any precipitation can be seen at this point, break the precipitation by pipetting up and down 10 times.

9.Assemble a HiBind DNA Mini column in a 2 ml collection tube (provided). Transfer the entire sample from Step 10 into the column, including any precipitate that may have formed. Centrifuge at 10000 x g for 1 min to bind DNA. Discard the collection tube and filtrate.

10.Place the column into a second 2 ml tube and wash by adding 500 μl Buffer HB. Centrifuge at 10000 x g for 1 min. Discard flow-through and reuse the collection tube.

11.Place the column into the same collection tube and wash by adding 700 μl DNA Wash Buffer diluted with ethanol. Centrifuge at 10000 x g for 1 min. Discard flow-through and reuse the collection tube.

NOTE: DNA Wash Buffer is supplied as a concentrate and must be diluted with absolute ethanol according to the instructions on Page 4, under “Before Starting”.

12.Wash thee clumn with a second 700 μl DNA Wash Buffer and centrifuge as above. Discard flow-through and reuse the collection tube.

13.Using the same 2 ml collection tube, centrifuge HiBind DNA Mini Column at maxi speed for 2 min to dry the column. This step is critical for removal of trace ethanol that might otherwise interfere with down stream applications.

14.Place the column into a nuclease-free 1.5 ml microfuge tube and add 50-100 μl of preheated (65℃) Elution Buffer to HiBind DNA Mini column matrix. Allow columns to incubate for 3 to 5 min at room temperature after addition of Elution Buffer.

NOTE: Incubating the HiBind DNA column at 65℃ rather than at room temperature prior to centrifugation will give a modest increase in DNA yield per elution.

15.To elute DNA from the column, centrifuge at 10000 x g for 1 min.

16.Repeat the elution with a second 50-100 μl Elution Buffer.

Sichuan university