Buccal Swab /FTA Card DNA Extraction Kit

Composition

Storage

1.      Proteinase K can be transported at room temperature. After receiving the product, please store at -20℃.

2.      Other reagents and components can be stored for up to two years at room temperature (0-30°C). For longer storage, it is recommended to keep at 2-8℃.(the product stored at 2~8℃ should be restored to room temperature before use).

Introduction

This product is suitable for the extraction of total DNA (including genomic DNA, mitochondrial DNA and possibly viral DNA) from buccal swabs. The released DNA is bound to the Spin Column, the degraded proteins and PCR inhibitors are filtered out, and the DNA is washed by Buffer WA and Buffer WB and eluted with Buffer TE, which can be used in various molecular biology experiments.

Equipment and Reagents to Be Supplied by User

1.      Absolute ethanol

2.      1.5 ml and 2 ml microcentrifuge tube

3.      Pipettes and tips

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

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

6.      Thermostatic shaker or water bath and vortexer

Preparation Before Use

1.      If the centrifuge has a refrigeration function, set the temperature to 25℃.

2.      Set the thermostatic shaker or water bath temperature to 56℃ and 70℃, and incubate the Buffer AT and Buffer TE to 56℃.

3.      Add absolute ethanol to Buffer WA and Buffer WB according to the instructions on the bottle label and tick the box on the label to mark "Ethanol Added".

Protocol

1.     Cut off the swab part of the buccal swab and place it into a 2 ml microcentrifuge tube.

* Sampling method: Wipe the swab inside the buccal 10 times on each side of the cheek.

* Do not eat or drink within half an hour before taking the sample.

2.     Add 400 µl Buffer AT preheated at 56℃, then add 20 µl Proteinase K, vortex for about 15 sec to mix well.

3.     Incubate for 1 h at 56℃ in a thermostatic shaker at 900 rpm.

* If incubate at a 56℃ water bath for 1 h, vortex several times every 15 min during the water bath to aid lyse.

4.     Add 400 µl Buffer SL and vortex for about 15 sec to mix well. Incubate for 10 min at 70℃ in a thermostatic shaker at 900 rpm.

* If incubate at a 70℃ water bath for 10 min, vortex several times every 3 min during the water bath to aid lyse.

5.     Add 200 µl absolute ethanol and vortex for about 15 sec to mix well. Spin down the solution on the cap settles to the bottom of the tube.

6.     Transfer 700 µl of the supernatant from Step 5 to a Spin Column (the Spin Column is placed in a 2 ml Collection Tube). Close the lid and centrifuge at 12000 rpm for 30 sec.

* Be careful not to dip the solution onto the nozzle of Spin Column. Otherwise, it cannot completely wash the Spin Column in subsequent washing steps.

7.     Discard the filtrate, 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. To avoid contamination of the centrifuge by the filtrate adhering to the mouth of the Collection Tube, invert the 2 ml Collection Tube and slap once on a paper towel.

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

8.     Discard the filtrate, 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 into Buffer WB.

9.     Discard the filtrate, place the Spin Column back into the 2 ml Collection Tube and centrifuge at 14000 rpm for 1 min.

* If the centrifuge does not reach 14000 rpm, centrifuge at full speed for 2 min.

* Do not omit this step, or the purified DNA maybe mixed with ethanol and maybe inhibit the subsequent PCR.

10.  Discard 2 ml Collection Tube, place the Spin Column in a clean 1.5 ml microcentrifuge tube, add 60-100 µl Buffer TE preheated at 56℃ into the Spin Column, close the lid, stand at room temperature for 1 min, and centrifuge at 12000 rpm for 30 sec.

* If the centrifuge does not have a leak-proof cap, change the centrifuge condition to 8000 rpm for 1 min to avoid damage to the centrifuge.

11.  Discard the Spin Column, and the eluted DNA can be immediately used in various molecular biology experiments or stored at -20℃ for later use.