Circulating Nucleic Acid Isolation Kit

Circulating Nucleic Acid Isolation Kit Instructions

Composition

Storage

1.      Proteinase K and Carrier RNA can be transported at room temperature and stored at -20℃.

2.      If other reagents and items are stored at room temperature (0~30℃), they can keep their performance unchanged for 2 years, and if the product is stored at 2~8℃, the validity period can be extended to more than 2 years (the product stored at 2~8℃ should be restored to room temperature before use).

Introduction

Circulating Nucleic Acid Isolation  Kit  is suitable for exacting and purifying circulating nucleic acids from 1-5 ml fresh or frozen body fluid samples, including plasma, serum, urine, CSF, and cell culture supernatants. The exaction protocol specifically designed for large volumes of plasma (5 ml) ensures that very trace amounts (1-2 copies/ml) of nucleic acids can also be efficiently concentrated and adsorbed onto the spin column, and the trace amounts of nucleic acids are washed with Buffer WA, Buffer WBR, and eluted by Buffer TE, which can be used in various molecular biology experiments.

Equipment and reagents to be supplied by users

1．  Absolute ethanol, isopropanol

2．  50 ml tubes and 1.5 ml tubes (DNase-free & RNase-free 1.5 ml tubes recommended)

3．  Pipette tips (to avoid contamination between samples, use DNase-free & RNase-free tips with filters)

4．  Disposable gloves and protective equipment and tissues

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

6．  Water bath with vortexer

Preparation before use

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

2.        Set the water bath temperature to 60℃ and incubate Buffer TE to 60℃.

3.        Add isopropanol to Buffer AC and absolute ethanol to Buffer WA and Buffer WBR according to the instructions on the label of the reagent bottle, tick the box on the label to mark "Isopropanol Added" and "Ethanol Added".

Protocol

The following steps are designed for the exaction of nucleic acids from 5 ml plasma, and if nucleic acids are exacted from other volumes of plasma, reagents are added in the ratio of 1 ml plasma + 100 μl proteinase K + 1 ml Buffer VL + 2 ml Buffer AC, while the dosage of Carrier RNA, Buffer WA, Buffer WBR, and Buffer TE remains the same.

1.     Add 500 μl Proteinase K and 5 ml plasma sample into a 50 ml centrifuge tube (not provided).

* Try to use freshly isolated or freeze-thaw samples no more than once for nucleic acid isolation and exaction. When using plasma or serum for nucleic acid isolation with more than one freeze-thaw cycle, the plasma or serum should be centrifuged at 6800 rpm for 3 min, and the supernatant should be aspirated for nucleic acid isolation, otherwise the condensed protein generated by repeated freeze-thaw in plasma or serum may block the spin column during the operation, resulting in nucleic acid isolation failure.

* Some nucleic acid fragments used for testing have only a few copies per milliliter of sample, so the maximum sample volume possible should be used for nucleic acid concentration and exaction, where possible.

2.     Add 5 ml Buffer VL and 10 μl Carrier RNA, vortex for about 30 sec to mix.

* Do not add proteinase K directly to Buffer VL.

3.     Incubate the centrifuge tube at 60℃ for 30 min.

4.     Add 10 ml Buffer AC and mix well.

* Ensure that isopropanol has been added to Buffer AC.

5.     Insert the connection tube into the socket of the negative pressure device, then insert the spin column on the connection tube, and finally insert the extension tube of the spin column on the spin column (see the instructions of the negative pressure device for details). Transfer the mixture from step 4 into the extension tube of the spin column, turn on the negative pressure, and let the liquid all filter through the spin column.

* To avoid cross-contamination, please do not reuse the connection tube.

* To avoid cross-contamination, do not insert the spin column directly into the socket of the negative pressure device.

6.     Turn off the negative pressure, remove and discard the extension tube. Add 600 μl Buffer WA to the spin column and turn on the negative pressure to aspirate the solution in the spin column.

* Ensure that absolute ethanol has been added to Buffer WA

7.     Turn off the negative pressure, add 800 μl Buffer WBR to the spin column, and turn on the negative pressure to aspirate the solution in the spin column.

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

8.     Turn off the negative pressure, add 900 μl absolute ethanol to the spin column, and turn on the negative pressure to aspirate the solution in the spin column.

9.     Turn off the negative pressure, wait for the negative pressure to disappear, remove and discard the connection tube, place the spin column into the 2 ml collection tube, close the lid, and centrifuge at 14,000 rpm for 1 min.

* If the centrifuge does not reach 14,000 rpm, centrifuge at its full speed for 2 min.

* Do not omit this step, otherwise the subsequent PCR effect may be affected due to the ethanol mixed in the purified nucleic acid.

10.  Discard the 2 ml tube, place the spin column in a 1.5 ml collection tube, add 50 μl Buffer TE to the spin column center, close the lid, incubate at room temperature for 3 min, and centrifuge at 12,000 rpm for 30 sec.

* Eluting nucleic acids with an elution volume of less than 50 μl does not ensure an increase in the concentration of nucleic acids but may reduce the elution efficiency of nucleic acids because the membrane is not fully wetted. In the 50 μl PCR reaction system, 20 μl nucleic acid purified with this product was added as a template, and no obvious inhibition reaction was observed. It is recommended to increase the amount of template used to increase the sensitivity of the detection.

11.  Discard the spin column, the eluting circulating nucleic acids can be used immediately for PCR or RT-PCR testing or stored at -20℃ or lower for later use.