基本描述

英文名称

Magbead Free-Circulating DNA Maxi Kit

储存温度

2-8°C储存,室温

运输条件

冰袋运输

产品介绍

产品内容

M670148	Component	2 mL×48 T	Storage
M670148A	Buffer MPL	120 mL	RT
M670148B	20% SDS	6 mL	RT
M670148C	Buffer GW1 (concentrate)	80 mL	RT
M670148D	Buffer GCW2 (concentrate)	40 mL	RT
M670148E	RNase-Free Water	30 mL	RT
M670148F	Proteinase K	2×1.25 mL	RT
M670148G	Magbeads ZN	2×1 mL	2-8℃

产品简介

该试剂盒适用于从血浆、血清、羊水等无细胞体液中纯化回收游离 DNA （Free-circulating/Cell-free DNA）。高盐时，游离DNA结合于硅基包被的磁珠表面。漂洗后，游离DNA洗脱于RNase-Free Water 中。游离DNA的得率与样品类型、储存条件、时间以及个体间差异有很大关系。纯化得到的游离DNA质量稳定、可靠，可用于定量PCR、产前诊断等下游实验。

自备仪器、试剂

1 ．全自动核酸提取仪

2 ．无水乙醇、异丙醇

3 ．24 DW Plate and Tips Pack

实验前准备及重要注意事项

1 ．第一次使用前应按试剂瓶标签说明先在Buffer GW1中加入无水乙醇。 2 ．第一次使用前应按试剂瓶标签说明先在Buffer GCW2中加入无水乙醇。 3 ．若手动操作，在实验开始前将恒温混匀仪预热至60℃。

4 ．Magbeads ZN严禁冰冻和高速离心，否则可能会对Magbeads ZN造成不可逆的损害。 Magbeads ZN每次使用时请充分振荡混合均匀。

5. 用前请先检查Buffer MPL 和 20% SDS是否出现结晶或沉淀，如有结晶或沉淀现象，可在37°C水浴几分钟，即可恢复澄清。

操作步骤（手动，以2mL血浆为例）

1 ．按下表向离心管中依次加入30 μL Proteinase K 、2 mL血浆、 100 μL 20% SDS ，放于60℃、 1200 rpm恒温混匀仪上震荡20分钟，孵育结束后将离心管冰浴5-10 分钟。

注：如无恒温混合仪，将离心管涡旋震荡10秒钟后放于60℃水浴锅中孵育20分钟，期间每隔7分

钟涡旋震荡10秒钟。

为避免蛋白酶K失活，请按下表试剂顺序依次加入，请勿将SDS直接加到蛋白酶 K溶液中。

Regent	血浆体积	血浆体积	血浆体积	血浆体积
Regent	1 mL	2 mL	4 mL	10mL
Proteinase K	15μL	30μL	60μL	150μL
血浆样本	1 mL	2 mL	4 mL	10mL
20% SDS	50μL	100μL	200μL	500μL
总体积	1.065mL	2.13 mL	4.26mL	10.65mL

2 ．孵育过程中，按下表准备裂解液/磁珠混合液，并混合均匀。

Regent	血浆体积	血浆体积	血浆体积	血浆体积
Regent	1 mL	2 mL	4 mL	10 mL
Buffer MPL	1 mL	2 mL	4 mL	10 mL
异丙醇	0.25 mL	0.5 mL	1 mL	2.5 mL
Magbeads ZN	15 μL	30 μL	60 μL	150 μL
总体积	1.265 mL	2.53 mL	5.06 mL	12.65 mL

3 ．将步骤2中准备的裂解液/磁珠混合液加到步骤1样本管中。涡旋震荡1分钟，然后手工上下颠倒或使用混匀仪混匀5-10分钟，使磁珠一直处于悬浮状态。

4 ．将离心管放于磁力架上静置，待磁珠吸附到磁力架上，管内溶液变澄清后，翻转离心管冲洗瓶盖上残留磁珠，再放置1分钟左右，之后弃去溶液。

5 ．向离心管中加入1 mL Buffer GW1（使用前请检查是否已加入无水乙醇），震荡混匀后将混悬液转移到新的1.5 mL离心管中。

6 ．将离心管固定于磁力架上静置1分钟，之后弃去溶液。

7 ．向离心管中加入1 mL Buffer GW1（使用前请检查是否已加入无水乙醇），涡旋震荡5秒钟后放于25℃、 1600 rpm的恒温混匀仪上震荡结合2分钟。

8 ．将离心管固定于磁力架上静置1分钟，之后弃去溶液。

9 ．向离心管中加入1 mL Buffer GCW2（使用前请检查是否已加入无水乙醇），涡旋震荡5秒钟后放于25℃、 1600 rpm的恒温混匀仪上震荡结合2分钟。

10．将离心管固定于磁力架上静置1分钟，之后弃去溶液。

11．重复步骤9-10。

12．离心管短暂离心后，将其重新固定于磁力架上用移液器去除管底溶液，开盖室温放置5-10分钟使乙醇充分挥发。

13．向离心管中加入50-100 μL RNase-Free Water后涡旋震荡使磁珠充分悬浮于洗脱

液中，之后将离心管固定于25℃、 1600 rpm的恒温混匀仪上震荡洗脱10分钟。

14．将离心管固定于磁力架上静置2分钟，待Magbeads完全吸附于离心管侧壁后用移液器将洗脱液转移至新的离心管中-20℃保存备用。

操作步骤（以4mL 血浆为例）

1 ．按下表向24 DW深孔板中加入试剂：

血浆体积	2 mL	4 mL
位置	试剂及用量	试剂及用量
Plate 1	Proteinase K：30 μL 血浆：2 mL 20% SDS：100 μL	Proteinase K：60 μL 血浆：4 mL 20% SDS：200 μL
Plate 2	Buffer GW1: 1 mL	Buffer GW1: 1 mL
Plate 3	Buffer GW1: 1 mL	Buffer GW1: 1 mL
Plate 4	Buffer GCW2: 1 mL	Buffer GCW2: 1 mL
Plate 5	Buffer GCW2: 1 mL	Buffer GCW2: 1 mL
Plate 6	RNase-Free Water: 100 μL	RNase-Free Water: 100 μL

2 ．将“24 DW Plate and Tips Pack”放入核酸提取仪的相应位置中，运行“CW2560 ctDNA程序”。

3 ．约25分钟后仪器暂停，将“Plate 1”拿出仪器后立即放到冰上5-10分钟，然后按下表加入试剂。

血浆体积	2 mL	4 mL
位置	试剂及用量	试剂及用量
Plate 1	Buffer MPL：2 mL 异丙醇： 0.5 mL Magbeads ZN：30 μL	Buffer MPL：4 mL 异丙醇： 1 mL Magbeads ZN：60 μL

4 ．将24 DW深孔板放回仪器中，继续运行程序。约45分钟后程序运行结束。把 “Plate 6”中的DNA洗脱产物转移至离心管中-20℃保存备用。

Product content

M670148	Component	2 mL×48 T	Storage
M670148A	Buffer MPL	120 mL	RT
M670148B	20% SDS	6 mL	RT
M670148C	Buffer GW1 (concentrate)	80 mL	RT
M670148D	Buffer GCW2 (concentrate)	40 mL	RT
M670148E	RNase-Free Water	30 mL	RT
M670148F	Proteinase K	2×1.25 mL	RT
M670148G	Magbeads ZN	2×1 mL	2-8℃

Product Introduction：
This reagent kit is suitable for purifying and recovering free circulating/cell free DNA from cell-free body fluids such as plasma, serum, and amniotic fluid. When high salt is present, free DNA binds to the surface of silicon coated magnetic beads. After rinsing, free DNA is eluted into RNase Free Water. The yield of free DNA is closely related to sample type, storage conditions, time, and individual differences. The purified free DNA has stable and reliable quality, and can be used for downstream experiments such as quantitative PCR and prenatal diagnosis.
Self provided instruments and reagents：
1. Fully automatic nucleic acid extractor
2. Anhydrous ethanol and isopropanol
3.24 DW Plate and Tips Pack
Preparation and important precautions before the experiment
1.Before the first use, anhydrous ethanol should be added to Buffer GW1 according to the instructions on the reagent bottle label.
2.Before the first use, anhydrous ethanol should be added to Buffer GCW2 according to the instructions on the reagent bottle label.
3. If manually operated, preheat the constant temperature mixer to 60 ℃ before the experiment begins.
4. Magheads ZN is strictly prohibited from freezing and high-speed centrifugation, otherwise it may cause irreversible damage to Magheads ZN. Please shake and mix Magheads ZN thoroughly and evenly every time you use it.
5. Before use, please check if there is any crystallization or precipitation in the Buffer MPL and 20% SDS. If there is any crystallization or precipitation, you can take a water bath at 37 ° C for a few minutes to restore clarity.
Operation steps (manual, using 2mL plasma as an example)
1. Add 30 to the centrifuge tube in sequence according to the table below μ L Protein K, 2 mL plasma, 100 μ L 20% SDS, shake on a constant temperature mixer at 60 ℃ and 1200 rpm for 20 minutes, and after incubation, ice bath the centrifuge tube for 5-10 minutes.
Note: If there is no constant temperature mixer, vortex the centrifuge tube for 10 seconds and incubate it in a 60 ℃ water bath for 20 minutes. During this period, vortex every 7 minutes for 10 seconds.
To avoid inactivation of protease K, please add the reagents in the order shown in the table below. Do not add SDS directly to the protease K solution.

Regent	plasma volume	plasma volume	plasma volume	plasma volume
Regent	1 mL	2 mL	4 mL	10mL
Proteinase K	15μL	30μL	60μL	150μL
Plasma samples	1 mL	2 mL	4 mL	10mL
20% SDS	50μL	100μL	200μL	500μL
Total Volume	1.065mL	2.13 mL	4.26mL	10.65mL

2.During the incubation process, prepare the lysis solution/magnetic bead mixture according to the table below and mix evenly.

Regent	plasma volume	plasma volume	plasma volume	plasma volume
Regent	1 mL	2 mL	4 mL	10 mL
Buffer MPL	1 mL	2 mL	4 mL	10 mL
isopropanol	0.25 mL	0.5 mL	1 mL	2.5 mL
Magbeads ZN	15 μL	30 μL	60 μL	150 μL
Total Volume	1.265 mL	2.53 mL	5.06 mL	12.65 mL

3. Add the cracking solution/magnetic bead mixture prepared in step 2 to the sample tube in step 1. Vortex for 1 minute, then manually invert or mix with a mixer for 5-10 minutes to keep the magnetic beads in a suspended state.
4. Place the centrifuge tube on the magnetic frame and let it stand. Wait for the magnetic beads to adsorb onto the magnetic frame. After the solution inside the tube becomes clear, flip the centrifuge tube to rinse the residual magnetic beads on the bottle cap. Leave it for about 1 minute, and then discard the solution.
5. Add 1 mL of Buffer GW1 to the centrifuge tube (please check if anhydrous ethanol has been added before use), shake and mix well, then transfer the suspension to a new 1.5 mL centrifuge tube.
6. Fix the centrifuge tube on a magnetic rack and let it stand for 1 minute, then discard the solution.
7. Add 1 mL of Buffer GW1 to the centrifuge tube (please check if anhydrous ethanol has been added before use), vortex for 5 seconds, and then place it on a constant temperature mixer at 25 ℃ and 1600 rpm for 2 minutes.
8. Fix the centrifuge tube on a magnetic rack and let it stand for 1 minute, then discard the solution.
9. Add 1 mL of Buffer GCW2 to the centrifuge tube (please check if anhydrous ethanol has been added before use), vortex for 5 seconds, and then place it on a constant temperature mixer at 25 ℃ and 1600 rpm for 2 minutes.
10. Fix the centrifuge tube on a magnetic stand and let it stand for 1 minute, then discard the solution.
11. Repeat steps 9-10.
12. After briefly centrifuging the centrifuge tube, fix it back on the magnetic frame and use a pipette to remove the solution at the bottom of the tube. Open the lid and let it sit at room temperature for 5-10 minutes to allow the ethanol to fully evaporate.

13. Add 50-100 to the centrifuge tube μ The vortex oscillation after L RNase Free Water causes the magnetic beads to fully suspend during elution
In the liquid, then fix the centrifuge tube on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and wash for 10 minutes.
14. Fix the centrifuge tube on a magnetic frame and let it stand for 2 minutes. After Magheads are completely adsorbed on the side wall of the centrifuge tube, transfer the eluent to a new centrifuge tube using a pipette and store it at -20 ℃ for later use.
Operation steps (using 4mL plasma as an example)
1. Add reagents to the 24 DW deep well plate according to the following table:

plasma volume	2 mL	4 mL
position	Reagent and dosage	Reagent and dosage
Plate 1	Proteinase K：30 μL plasma：2 mL 20% SDS：100 μL	Proteinase K：60 μL plasma：4 mL 20% SDS：200 μL
Plate 2	Buffer GW1: 1 mL	Buffer GW1: 1 mL
Plate 3	Buffer GW1: 1 mL	Buffer GW1: 1 mL
Plate 4	Buffer GCW2: 1 mL	Buffer GCW2: 1 mL
Plate 5	Buffer GCW2: 1 mL	Buffer GCW2: 1 mL
Plate 6	RNase-Free Water: 100 μL	RNase-Free Water: 100 μL

2. Place the "24 DW Plate and Tips Pack" in the corresponding position of the nucleic acid extractor and run the "CW2560 ctDNA program".
3.After about 25 minutes, the instrument will pause. Take out "Plate 1" and immediately place it on ice for 5-10 minutes, then add the reagent according to the table.

plasma volume	2 mL	4 mL
position	Reagent and dosage	Reagent and dosage
Plate 1	Buffer MPL：2 mL isopropanol ： 0.5 mL Magbeads ZN：30 μL	Buffer MPL：4 mL isopropanol ： 1 mL Magbeads ZN：60 μL

4. Put the 24 DW deep hole plate back into the instrument and continue running the program. The program will finish running in about 45 minutes. Transfer the DNA elution products from "Plate 6" to a centrifuge tube and store at -20 ℃ for future use.

安全和危险性（GHS）

SDS英文版

质量标准

质量标准

m670148_magbead free-circulating dna maxi kit.pdf

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

输入批号以搜索分析图谱:

溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

质量

=

浓度

x

体积

x

分子量

g/mol

稀释计算器

计算配制储备溶液所需的稀释度。

浓度1（C1）

x

体积 1 (V1)

=

浓度 2 (C2)

x

体积 2 (V2)

复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

=

质量（小瓶）

÷

所需的复溶浓度

磁珠法大体积游离DNA提取试剂盒

库存信息

基本描述

安全和危险性（GHS）

质量标准

质检证书（CoA,COO,BSE/TSE 和分析图谱)

溶液计算器

摩尔浓度计算器

稀释计算器

复溶计算器