Bacterial DNA Isolation Kit

Product Composition

Product Storage and Expiry Date

1．  Lysozyme can be transported at room temperature, please store at 2~8℃ after receiving the product.

2．  Other reagents and components are stored at room temperature (0 ~ 30 ℃), the performance of the product can be maintained without significant change within two years; if the product is stored at 2~8 ℃, the validity period of the product can be extended to more than two years (the product stored at 2~8℃ should be restored to room temperature before use).

Product Description

This product is suitable for the isolation of total DNA from 1-5 ml bacterial cultures.The bacteria are lysed by Buffer L1 after lysozyme wall-breaking treatment, and then precipitated by Buffer L2 to remove proteins and cellular debris, and genomic DNA in the centrifugation supernatant can be bound to the spin column. After washing with Buffer WA and Buffer WB to remove the protein and PCR inhibitors remaining on the membrane, the genomic DNA is eluted with Buffer TE and can be used immediately for various molecular biology experiments.

Equipment and Reagents to Be Supplied by User

1.        Deionised purified water and absolute ethanol.

2.        1.5 ml microcentrifuge tubes, pipettes and tips (pipette tips with filter cartridges are recommended to avoid cross-contamination between samples)

3.        Protective equipment such as disposable latex gloves and paper towels

4.        Microcentrifuge(s) (with rotor for 1.5 ml and 2 ml microcentrifuge tubes)

5.        Water bath and vortexer

Preparation Before Use

1.        If the centrifuge has refrigeration function, please set the temperature to 25°C.

2.        Set the temperature of the water bath to 37°C and incubate the Buffer TE at 37°C.

3.        Prepare 100 mg/ml Lysozyme solution according to the number of samples to be extracted at one time (100 µl Lysozyme solution per sample. e.g., for the extraction of bacterial genomic DNA from 6 samples, weigh 65 mg Lysozyme and add 650 µl deionised purified water to make 650 µl Lysozyme solution.）

Note: repeated freezing and thawing of lysozyme solution greatly affects its activity, if more lysozyme solution is prepared at one time, it should be divided into small portions and stored at -20℃, and any remaining lysozyme solution after thawing and use should be discarded and not frozen again.

4.        Add absolute ethanol to Buffer WA and Buffer WB according to the instructions on the label of the reagent bottle, and tick the box on the label to mark "Ethanol added".

Protocol:

1.       Collect 1-5 ml bacterial culture in a 1.5 ml microcentrifuge tube, add 200 µl Buffer TE and vortex to fully suspend the bacteria.

* Certain divalent cations can inhibit the activity of lysozyme, so if the bacterial culture contains divalent cations (e.g. MRS medium), an additional washing step should be performed after collecting the bacteria: add 1 ml deionised water, vortex to suspend the bacteria, then centrifuge at 12,000 rpm for 30 seconds, discard the supernatant, and then add 200 µl Buffer TE and vortex to fully suspend the bacteria.

* Some specific bacteria may change the pH of the medium after cultivation (e.g. Lactobacillus), which can inhibit the activity of lysozyme, it is also need to be washed once after collecting the bacteria in the same way as above.

* Methods of bacterial collection:

A.Bacteria in suspension culture: centrifuge at 12,000 rpm for 30 seconds to collect bacteria in 1-5 ml of bacterial culture, discard medium.

B. Single colonies in petri dishe: add 200 µl Buffer TE to a 1.5 ml microcentrifuge tube, colonies were scraped with an inoculating loop and eluted in Buffer TE.

2.       Add 100 µl Lysozyme solution, mix by vortexing for 15 seconds, and incubate at 37°C for 30-60 minutes.

* Most of the bacteria have been fully wall-broken after 30 minutes in the water bath, but some bacteria with thicker cell walls (e.g. Staphylococcus aureus) need to be incubated for 1-2 hours to be fully wall-broken. Please adjust the water bath time appropriately according to different categories of bacteria.

3.       Add 225 µl Buffer L1, close the lid, shake vigorously 3~5 times and vortex for 30 seconds.

* If DNA is extracted from freshly cultivated bacteria, some RNA from the bacteria may be isolated together, but the presence of RNA does not affect PCR-related experiments. If RNA needs to be completely removed, 4 µl RNase A (100 mg/ml, not provided in this kit) can be added in this step.

4.       Add 225 µl Buffer L2, close the lid, shake vigorously 3~5 times and vortex for 30 seconds.

5.       Centrifuge at 13000 rpm for 2 minutes.

6.       Decanting the supernatant from step 5 into a spin column (the spin column is placed in a 2 ml collection tube), close the lid and centrifuge at 12000 rpm for 30 seconds.

7.       Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube, add 500 µl Buffer WA to the spin column, close the lid and centrifuge at 12000 rpm for 30 seconds.

* The filtrate does not need to be completely discarded, if you want to avoid the contamination of the centrifuge by the filtrate adhering to the nozzle of the collection tube, you can slap the 2 ml collection tube upside down on a paper towel once.

* Make sure absolute ethanol has been added into Buffer WA.

8.       Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube, add 600 µl Buffer WB to the spin column, close the lid and centrifuge at 12000 rpm for 30 seconds.

* Make sure absolute ethanol has been added into Buffer WB.

9.       Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube and centrifuge at 14000 rpm for 1 minute.

* If the top speed could not reach 14000 rpm, centrifuge at top speed for 2 minutes.

* Do not omit this step, otherwise, it may cause problems in downstream applications due to the residual ethanol in the eluate.

10.    Discard the 2 ml collection tube, place the spin column in a clean 1.5 ml microcentrifuge tube, add 100~200 µl pre-warmed Buffer TE to the centre of the membrane, close the lid and incubate for 1 minute at room temperature, then centrifuge at 12000 rpm for 30 seconds.

* If the centrifuge does not have a leak-proof cap, please change the centrifugation condition to 8000 rpm for 1 minute to prevent the 1.5 ml microcentrifuge tube cover from falling off and damaging the centrifuge.

11.    Discard the spin column, the eluted DNA can be used immediately for various molecular biology experiments; or store the DNA at -20°C for later use.