Nucleic Acid Extraction Kit (Magnetic Bead Method) IFU

【Product Name】 【Storage and Validity】

Nucleic Acid Extraction Kit (Magnetic Bead Method) 1. Stored at 2 ～ 8℃ for 12 months, protecting from direct sunlight and moisture.
2. Stored at room temperature for 60 days once opened.
【Product Code】
A-100, A-200; B-100, B-200; T-200 【Applicable Instruments】
1. Manual operation: magnetic separator, centrifuge, dry bath.
【Packing Specifications】 2. Nucleic acid extraction system: automatic or semi-automatic nucleic acid
extraction system based on magnetic bead absorption principle.
Catalog No. Package Size Catalog No. Package Size
A100-32 32 T/Kit B100-32 32 T/Kit 【Sample Requirements】
1. Sample types: Liquid samples such as serum, plasma, nasopharyngeal
A100-96 96 T/Kit B200-8 8 T/Kit swab, cell preservation fluid , tissue fluid, urine and secretions.
A200-32 32 T/Kit B200-16 16 T/Kit 2. Sample collection: Collect sample via routine method.
3. Sample preservation and transportation: The samples collected can be
A200-96 96 T/Kit B200-32 32 T/Kit
used for nucleic acid extraction immediately, or stored at 2~8℃ (less than 24
B100-8 8 T/Kit T200-32 32 T/Kit hours), at -20℃ for long-term preservation. Prevent from freeze-thaw cycles.
B100-16 16 T/Kit T200-96 96 T/Kit The samples should be transported in sealed cooler box or styrofoam box
with ice seal.

【Intended Use】 【Test Method】

For the extraction, enrichment and purification of nucleic acid (DNA/RNA) in 1. Manual Operation (Take A-200 as example, Figure 1)
samples. Its processed products are for clinical in vitro diagnostics.

【Test Principle】
The magnetic Beads in the kit have specific polymeric groups of adsorbed
nucleic acid (DNA/RNA) on the surface.
In special conditions like hypersaline, cells or viruses in the samples lyse Extraction Magnetic Proteinase Working Add sample to Lyse at 55℃ Absorbed
rapidly and release nucleic acids, which are specifically adsorbed by Reagent I Beads K Solution Working Solution for 4 min
Solution
magnetic Beads. Nucleic acids on the magnetic beads will be separated Lysate: 500 μL [Working Solution]
from the liquid phase when the magnetic separator is used. Residual Pretreatment: 500 μL [Extraction + 200 μL sample, mix well, lyse
impurities and inhibitors in the liquid phase are removed by washing with Reagent I] + 4 μL [Magnetic Beads at 55℃ for 4 min, absorbed by
extraction reagent II. Finally, nucleic acids are eluded from the magnetic Solution] +15 μL [Proteinase K], mix magnetic separator for 1 min, and
beads by changing the liquid phase conditions, so as to separate nucleic into [Working Solution]. discard the supernatant.
acid rapidly and efficiently.

【Main Components】
Table 1 Main Components of A-100/A-200

Constituents A-100 A-200

Main Components
of Kit 32 T/Kit 96 T/Kit 32 T/Kit 96 T/Kit
Add Elute at 80℃ Absorbed Add [Extraction Absorbed
<50% Guanidinium [Elution Buffer] for 2 min Reagent II]
Isothiocyanate,
Extraction 8 mL 24 mL 8 mL 48 mL
<10% Tris Buffer,
Reagent I ×1 bottle ×1 bottle ×2 bottles ×1 bottle Elution: add 50~100 μL [Elution Washing: add 600 μL [Extraction
<50% Dimethyl
carbinol Buffer], elute at 80℃ for 2 min, Reagent II], mix well, absorbed by
absorbed by magnetic separator for magnetic separator for 1 min, and
Extraction 14.4 mL 43.2 mL 9.6 mL 60 mL <10% Tris Buffer, 30 s, reserve the supernatant. discard the supernatant.
Reagent II ×1 bottle ×1 bottle ×2 bottles ×1 bottle <10% NaCl

6.4 mL 19.2 mL 6.4 mL 19.2 mL Figure 1 Manual Operation of A-200

Elution Buffer Deionized water
×1 bottle ×1 bottle ×1 bottle ×1 bottle
1.1 Take out all the components in the kit, keep them at room temperature
Magnetic and mix them well to be ready for use. If there is a small amount of crystal in
64 μL 192 μL 128 μL 480 μL <50%
Beads
×1 tube ×1 tube ×1 tube ×1 tube Magnetic Beads [Extraction Reagent I], reagent cannot be used until it is fully dissolved.
Solution
1.2 500 μL [Extraction Reagent I] + 4 μL [Magnetic Beads Solution] + 15 μL
Proteinase K
320 μL 960 μL 480 μL 1440 μL <5% [Proteinase K] = Working Solution/T (A-100: 250 μL [Extraction Reagent I] +
×1 tube ×1 tube ×1 tube ×1 tube Proteinase K 2 μL [Magnetic Beads Solution] + 10 μL [Proteinase K] = Working Solution/T).
Instrument Add reagents and solutions proportionally and make them well-mixed. (Note:
12 mL 24 mL <20% [Working Solution] should be used within 30 minutes.)
system / /
×1 bottle ×1 bottle Mineral Oil
Solution 1.3 Add 500 μL [Working Solution] and 200 μL sample to a marked 1.5 mL
centrifuge tube. Shake and mix it for 5 s, then heat it for 4 min on a dry bath
Table 2 Main Components of B-100/B-200 at 55℃ . (A-100: 250 μL [Working Solution] + 100 μL samples.)
1.4 Centrifuge the tube for 5 s, place it on the magnetic separator for 1 min,
B-100 B-200 then discard the supernatant. (Note: try not to touch the magnetic beads on
Constituents
16 T 32 T 16 T 32 T Main Components the tube walls when discarding the supernatant.)
of Kit 8 T/Kit 8 T/Kit
/Kit /Kit /Kit /Kit 1.5 Add 600 μL [Extraction Reagent II] (A-100: Add 450 μL [Extraction
Reagent II]), cover the tube lid, shake and mix well for 5 s. Centrifuge
80 μL 160 320 120 240 480 it and place it on the magnetic separator for 1 minute, then discard the
﹤5%
Proteinase K ×1 μL×1 μL×1 μL×1 μL×1 μL×1
tube tube tube tube tube tube
Proteinase K supernatant. (Note: the same as step 1.4)
1.6 Remove the residual liquid at the bottom of the tube after one-minute standing.
Nucleic Acid 1.7 Add 50~100 μL [Elution Buffer], cover the tube lid, shake and mix well for
Extraction Reagent I,
Extraction
Reagent
4T 8T 16 T 4T 8T 16 T Extraction Reagent II, 5 s, and short spin.
×2 ×2 ×2 ×2 ×2 ×2 Elution Buffer, 1.8 Place the centrifuge tube on a dry bath at 80℃ , heat for 2 min.
Prepackaged
plates plates plates plates plates plates Magnetic Beads 1.9 Place the centrifuge tube on the magnetic separator, and take out
in 96-Well
Solution
Plates supernatant for following operation.

Table 3 Main Components of T-200 2. Operation of Semi-Automatic Nucleic Acid Extraction System
2.1 For B-100/B-200：
Constituents of Kit 32 T/Kit 96 T/Kit Note

Extraction Reagent I 32 T×1 plate 96 T×1 plate

Extraction Reagent II 32 T×1 plate 96 T×1 plate

Applicable to
Elution Buffer 32 T×1 plate 96 T×1 plate automated nucleic
acid extraction Proteinase k 96-well plates Sample 96-well plates
Magnetic Beads equipment
32 T×1 plate 96 T×1 plate Add 15μL [Proteinase K] (B-100: 10μL)
Solution
to the position A1~H1 and A7~H7 in Add 200 μL sample (B-100: 100μL) in
Proteinase K 480 μL×1 tube 1440 μL×1 tube order order
 Nucleic Acid Extraction Kit (Magnetic Bead Method) IFU

experiments such as PCR.

e.g.: 200 μL diluted HBV reference material (10 IU/mL) from WHO (NIBSC
code: 10/264) was extracted by the kit to get 50 μL analyte. The analyte
was detected by HBV detection kit for 10 times. Positive rate was 100%,
as shown in Figure 2-1. 200 μL diluted HCV reference material (25 IU/mL)
(5th WHO International Standard for HCV NAT, NIBSC code: 14/150) was
96-well plates Semi-automatic nucleic
extracted by the kit to get 50 μL analyte. The analyte was detected by HCV
acid extraction system detection kit for 10 times. Positive rate was 100%, as shown in Figure 2-2;
e.g.: DNA Pseudoviridae with a concentration of 5×108 IU/mL was diluted
Load the 96-well plate to instrument
with negative serum to 5×107 IU/mL, 5×106 IU/mL, 5×105 IU/mL, 5×104 IU/
Figure 2 Semi-Automatic Operation of B-200 mL, 5×103 IU/mL, 5×102 IU/mL and 50 IU/mL. They were extracted by the kit
and the analytes were detected, the results were shown in Figure 2-3; The
2.1.1 Take out all the components in the kit, keep them at room temperature RNA Pseudoviridae with a concentration of 5×107 IU/mL was diluted with
to be ready for use. Dump the liquid that adheres to the aluminum film and negative serum to 5×106 IU/mL, 5×105 IU/mL, 5×104 IU/mL, 5×103 IU/mL,
the well wall of the 96-well plates to the bottom of the plates. Let them stand 5×102 IU/mL, and 50 IU/mL. They were extracted by the kit and the analytes
for 3-5 minutes. were detected, the results were shown in Figure 2-4.
2.1.2 Carefully open the aluminum film of the 96-well plates, and add 15 μL
[Proteinase K] (B-100: 10 μL) to the position A1~H1 and A7~H7 in order,
then add 200 μL sample (B-100: 100 μL) in order.
2.1.3 Turn on the nucleic acid Extraction system, enter the page < Program
Edit >, and set the extraction process according to table 4:
Table 4 Running Program Setting
Absorption
Waiting Mixing
Magnetic Mixture Temperature Temperature
No. Position Name Time Time Volume
Beads Velocity State (℃)
(min) (min)
Time(sec)
1 2 Move 0 0 30 Slow 150 Closed 0 Figure 2 -1 Amplification Curve of Figure 2-2 Amplification Curve of
HBV Reference Material (10 IU/mL) HCV Reference Material (25 IU/mL)
Heating for
2 1 Lysis 0 4 60 Slow 500 55
Lysis
3 3 Wash 0 1 60 Slow 600 Closed 0
Heating for
4 6 Elution 0 2 30 Slow 50 80
Elution
5 1 Move 0 0 0 Slow 300 Closed 0
(Note: The parameters of volume is set according to the actualextraction
effect, may not be the same with reagent volumes.)
2.1.4 Click “Start” to run the extraction program. The process takes about
10 minutes. Figure 2-3 Amplification Curve of Figure 2-4 Amplification Curve of
2.1.5 Take out 96-well plates and pipette inventory nucleic acid solution from DNA Pseudoviridae RNA Pseudoviridae
the position A6-H6 and A12-H12 into 1.5 mL centrifuge tube for following
Figure 2 PCR Amplification Curve of Extract
operation. (a small amount magnetic beads could be removed by centrifuge
or magnetic separator.)
【Warnings and Precautions】
2.2 For T-200: 1. The components of the kit needs to be mixed well before use. If there is
2.2.1 Preparation: Data cable, Computer, Extraction program, Nucleic acid a small amount of crystal in [Extraction Reagent I], reagent cannot be used
extraction kit(T-200). until it is fully dissolved.
2.2.2 Connect instrument and computer, then import the extraction 2. Prevent sample from freeze-thaw cycles, mixed well before use.
program:Home- Connet- Transfer...- Upload- Chose the program- Change Otherwise, the concentration of extracted DNA/RNA will decrease.
the name “zhong.yuan” to “zhongyuan” (remove “.” ), import into folder. 3. Considering [Extraction Reagent I] contains flammable components,
2.2.3 Adding sample please keep away from fire sources or other risk factors.
Add 15 μL [proteinase K] and 200 μL sample to [Extraction reagent I]. 4. The disposal of waste liquid should be in accordance with local laws and
2.2.4 Instrument operation regulations.
Chose the extraction program “zhongyuan”- Click “Start”- Loading
reagents： 【References】
· Put [Elution buffer] plate to position 4; 1. Tang Y J，Zou J，Ma C，et al. Highly Sensitive and Rapid Detection of
· Put [Extraction Reagent II] plate to position 3; Pseudomonas aeruginosa Based on Magnetic Enrichment and Magnetic
· Put [Extraction Reagent I] plate to position 2; Separation.Theranostics,2013, 3(2):85-92.
· Put [Magnetic Beads Solution] plate to position 1, and then put the 2. Shain E B，Clemens J M. A new method for robust quantitative and
magnetic rod sleeve into the [Magnetic Beads Solution] plate. qualitative analysis of real-time PCR. Nucleic acids Res，2008，36(14):57-63.
Note：The placement of the 96-well plates are as shown below： 3. Li J M. Real-time Fluorescent PCR Technology [M] . Beijing: People's Military
Medical Press, 2007.
4
5
3

【Explanations on Symbols】

Symbol Explanation Symbol Explanation

IN VITRO DIAGNOSTIC CONSULT INSTRUCTIONS

MEDICAL DEVICE FOR USE

BATCH CODE USE-BY DATE

After the end of running, take out the corresponding 96-well plate( [Elution
buffer] plate) for subsequent experiment according to the instrument prompt. CATALOGUE NUMBER TEMPERATURE LIMIT

3. Operation of Automatic Nucleic Acid Extraction System MANUFACTURER EUROPEAN CONFORMITY

3.1 Take out all the components in the kit, keep them at room temperature
AUTHORIZED REPRESENTATIVE IN THE EUROPEAN COMMUNITY
and mix them well to be ready for use. If there is a small amount of crystal in
[Extraction Reagent I], reagent cannot be used until it is fully dissolved.
3.2 Refer to the operation manual and Standard of Operation of the 【 Manufacturer Information】
automatic nucleic acid extraction system to complete the extraction of Zybio Inc.
nucleic acid. Floor 1 to Floor 4, Building 30, No.6 of Taikang Road, Block C of Jianqiao Industrial Park,
Dadukou District, Chongqing, China 400082
Web: www.zybio.com
【Limitations on Test Method】 E-mail: info@zybio.com
This product needs to be used with a magnetic separator in manual Tel: +86(0)23 6865 5509
operation. Fax: +86(0)23 6869 9779

【Product Performance Index】 【 EC Representative】

1. Recovery Rate: ≥90%. Shanghai International Holding Corp. GmbH (Europe)
2. The extracted analyte can be directly used in molecular biology Eiffestrasse 80, 20537 Hamburg, Germany