Plant DNA Isolation Kit

Product Composition

Storage and Expiry Date

The kit can be stored at room temperature（0-30℃）for up to 2 years without showing any reduction in performance and would be stable more than 2 years if stored at 2-8℃ (Products stored at 2~8 °C should be restored to room temperature before use).

Product Introduction

This product is suitable for DNA extraction from biological samples that cannot be completely lysed, so it is most suitable for isolation and purification of DNA from plant, fungal, arthropods, etc. The specially designed buffer and spin column efficiently adsorb only large fragments of genomic DNA, so RNA can be removed in most cases without additional RNase digestion. The operation steps are simple and fast, only need to lyse and centrifuge the insoluble matter in the precipitated sample, the supernatant containing DNA can be added to the spin column for DNA adsorption, and the DNA in the spin column is washed by Buffer WA and Buffer WB and eluted in Buffer TE. The high-purity genomic DNA obtained is suitable for various molecular biology experiments.

Equipment and Reagents to Be Supplied by User

1．  Absolute ethanol

2．  Liquid nitrogen and mortar

3．  1.5 ml microcentrifuge tube, pipette, and tips

4．  Protective equipment such as latex gloves, disposable masks, and paper towels

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

6．  Vortexer and water bath

Preparation Before Use

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

2．Set the water bath temperature to 37℃ and incubate Buffer PL and Buffer TE to 37℃.

3．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.       Add liquid nitrogen to immerse the tissue, first grind about 300~500 mg (100~200 mg dry tissue) sample into fine particles, wait for liquid nitrogen to evaporate, and then quickly grind the tissue particles to powder. Weigh 50~100 mg (10~20 mg dry tissue) powdered tissue with a liquid nitrogen precooled 1.5 ml microcentrifuge tube.

* In order to grind the tissue into the smallest possible particles, and to avoid the ground tissue powder being difficult to weigh due to melting, the sample can be repeated grinding with multiple re-addition of liquid nitrogen. Plant tissue must be ground to powder form (as fine as flour) to adequately destroy the cell walls, otherwise the efficiency of final DNA recovery will be seriously affected.

* For plant tissues with low fiber content, weigh 100 mg sample and put into a mortar, add 100 μl of Buffer PL incubated at 37°C and ground at room temperature to homogenate, then add 400 μl Buffer PL incubated at 37°C to mix, all homogenates (including the resulting foam) are transferred to a clean 1.5 ml microcentrifuge tube, incubate at room temperature for 10 minutes. Continue with step 3.

2.       Add 500 μl Buffer PL incubated at 37℃, vortex for 30 sec to mix well, and incubate at room temperature for 10 min. Shake the microcentrifuge tube vigorously several times every 2~3 minutes during the incubation to aid the release of DNA.

3.       Add 350 μl Buffer K, close the lid, shake vigorously for 15 sec, and vortex for 30 sec. Centrifuge at 13000 rpm for 5 min.

4.       Transfer the supernatant from step 3 into a spin column (spin column placed in a 2 ml collection tube), close the lid and centrifuge at 12000 rpm for 30 sec.

5.       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 sec.

* The filtrate does not need to be completely discarded, if you want to avoid 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.

* Ensure that absolute ethanol has been added to Buffer WA.

6.       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 sec.

* Ensure that absolute ethanol has been added to Buffer WB.

* Some plant pigments may remain on the membrane of the spin column; in which case an additional washing step of absolute ethanol can be added (add 600 μl of absolute ethanol and centrifuge at 12000 rpm for 30 sec).

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

* 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.

8.       Discard the 2 ml collection tube, place the spin column in a clean 1.5 ml microcentrifuge tube, add 100~200 μl Buffer TE incubated at 37℃ to the center of the column membrane, close the lid, incubate for 2 min at room temperature, and centrifuge at 12000 rpm for 30 sec.

* If the centrifuge does not have a leak-proof lid, change the centrifugation conditions to 8000 rpm for 1 minute to avoid the tube cap falling off and damaging the centrifuge.

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