Yeast DNA Isolation Kit

Yeast DNA Isolation  Kit Instructions

Product Composition

Product storage

1.        Proteinase K storage solution and RNase A can be transported at room temperature. After receipt, please store the Proteinase K storage solution at -20°C and RNase A at 2~8°C.

2.        If the kit is stored at room temperature (0~30°C), its performance will remain unchanged for two years; if the product is stored at 2~8°C, the validity period of the product can be extended to more than two years.

Product Description

Purifying total DNA from 5 to 10 ml of yeast cell culture medium . Yeast cells are frozen in liquid nitrogen and then grinded to break the cells. Buffer AT and proteinase K storage solution are added to release the genomic DNA, and then Buffer K is used to precipitate proteins, polysaccharides and other impurities in the yeast cells. After adding the supernatant containing DNA to the nucleic acid purification column, the DNA is bound to the purification column, and the remaining proteins and PCR inhibitors are filtered out. After the DNA is washed with Buffer WA and Buffer WB, it is eluted with Buffer TE and can be used. for various molecular biology experiments.

Reagents and items that users need to prepare by themselves

1．  Anhydrous ethanol

2．  Liquid nitrogen, mortar or sample lysis tube M ( Simgen, Cat . No.: C-001-2)

3．  1.5 ml centrifuge tube , pipette and tip

4．  Latex gloves, disposable masks and other protective equipment and tissues

5．  Desktop small volume centrifuge (can be equipped with a rotor for centrifuging 1.5 ml centrifuge tubes and 2 ml centrifuge tubes)

6．  Vortex oscillator

7．  Water bath

Preparation Before use

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

2.        Set the water bath temperature to 70°C and incubate Buffer AT and Buffer TE to 70°C.

3.        Add absolute ethanol to Buffer WA and Buffer WB according to the instructions on the label of the reagent bottle, and check the box on the label to mark "Ethanol has been added".

Steps :

1.       Collect 5~10 ml of yeast cultured overnight and discard the supernatant .

* If you use a 1.5 ml centrifuge tube to collect yeast, you can centrifuge at 12,000 rpm for 30 seconds multiple times to enrich the bacteria.

2.       Add 100 µl of Buffer AT preheated at 70°C. Do not discard the pipette tip . Use the pipette tip to pipette the bacterial pellet directly, suck it out and transfer it to the mortar. Pour in liquid nitrogen to submerge the bacteria liquid (the bacteria liquid will immediately agglomerate into clumps when it hits the liquid nitrogen), and grind vigorously until the bacteria clumps become powdery.

* If the bacterial clumps cannot be ground into powder, liquid nitrogen should be added to continue grinding, otherwise the final DNA recovery efficiency will be seriously affected.

* In the absence of liquid nitrogen, you can directly grind for about 15 minutes to fully destroy the cell wall (if the liquid is evaporated during this process, add 100 µl Buffer AT to continue grinding) or use a sample lysis tube with the best grinding effect M ( Simgen, Cat . No.: C-001-2) destroys cell walls . For details, please see the article " The Effect of Different Grinding Methods on Yeast DNA Yield " on the simgenbio WeChat public account .

3.       When the powdered yeast begins to melt, add 500 µl Buffer AT and 2 µl RNase A to the mortar, continue grinding several times, use a pipette to transfer the lysate into a 1.5 ml centrifuge tube, and incubate in a 70°C water bath for 5 minutes .

* If the lysate is less than 500 µl, add Buffer AT to 500 µl.

4.       Add 20 µl Proteinase K stock solution, vortex for 30 seconds to mix, and place in a 70°C water bath for 20 minutes. During the water bath, turn the centrifuge tube several times every 2 to 3 minutes to help release the DNA.

5.       Add 350 µl Buffer K, cap the tube, shake vigorously for 15 seconds, and vortex for 30 seconds to mix. Centrifuge at 13,000 rpm for 5 minutes.

6.       Pour the centrifugation supernatant in step 5 into the nucleic acid purification column (the nucleic acid purification column is placed in a 2 ml centrifuge tube), cover the tube, and centrifuge at 12,000 rpm for 30 seconds.

7.       Discard the filtrate in the 2 ml centrifuge tube, place the nucleic acid purification column back into the 2 ml centrifuge tube, add 500 µl Buffer WA to the nucleic acid purification column, cap the tube, and centrifuge at 12,000 rpm for 30 seconds.

* There is no need to discard the filtrate completely. If you want to avoid contamination of the centrifuge by the filtrate adhering to the mouth of the centrifuge tube, place the 2 ml centrifuge tube upside down on a paper towel and tap it once.

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

8.       Discard the filtrate in the 2 ml centrifuge tube, place the nucleic acid purification column back into the 2 ml centrifuge tube, add 600 µl Buffer WB to the nucleic acid purification column, cap the tube, and centrifuge at 12,000 rpm for 30 seconds.

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

9.       Discard the filtrate in the 2 ml centrifuge tube, place the nucleic acid purification column back into the 2 ml centrifuge tube, cover the tube, and centrifuge at 14,000 rpm for 1 minute.

* If the centrifuge speed cannot reach 14000 rpm, centrifuge at the highest speed for 2 minutes.

* Do not omit this step, otherwise the purified nucleic acid may be mixed with ethanol, which may affect subsequent experimental results.

10.    Discard the 2 ml centrifuge tube, place the nucleic acid purification column in a clean 1.5 ml centrifuge tube, add 80~100 µl 70°C preheated Buffer TE to the purification column, cover the tube cap, and let stand at room temperature for 2 minutes, 12000 Centrifuge at rpm for 1 minute.

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

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