计算溶液所需的质量、体积或浓度。
,适用于对基线干扰要求严格的色谱和分析工作流程。
室温。常规运输 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
产品内容:
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该试剂盒提供了一种简单、快速、高效的血液DNA提取方法,适用于从新鲜或冷 冻抗凝血(柠檬酸盐、 EDTA、肝素处理过的血液样品)中提取血液DNA。在高盐存在 时, DNA结合于硅基包被的Magbeads表面。漂洗后,高纯度的DNA被洗脱于Buffer EB或去离子水中。 DNA得率与样品的类型,储存条件、时间以及样品中白细胞的含量 有很大关系,从200 μL 冷冻保存的抗凝血中通常可以提取得到3-10 μg基因组DNA。纯 化得到的DNA纯度好(A260/280 的比值在1.7-1.9之间, A260/230 的比值大于1.5),完整度 高(最高可达50 kb),可用于二代测序、克隆、定量PCR、芯片检测等下游实验。
该试剂盒可与液体工作站和磁棒法磁珠自动提取系统配套使用,简单、快速地进 行大规模提取,大大降低了实验者的工作量和实验中的人为误差。
自备仪器、试剂
1 .手动单管提取:
1)恒温混匀仪
2)2/15 mL磁力架
3)异丙醇、无水乙醇
2 .手动96孔深孔板提取:
1)恒温混匀仪
2)废液抽吸系统
3)手动连续分液器
4)电动连续分液器
5)手动连续分液器分液管(25 mL)
6)96孔板磁力架
7)异丙醇、无水乙醇
3 .磁棒法磁珠自动提取系统:
1)磁棒法磁珠自动提取系统
2)异丙醇、无水乙醇
实验前准备及重要注意事项
1.异丙醇与Magbeads混合物的制备(以制备提取10个样品所需量为例)
1)向合适容量(加入异丙醇和Magbeads后总体积小于离心管容积的2/3)的离心 管中加入3.3 mL【0.3×(10+1)=3.3】的异丙醇。
注意:如用移液器加入异丙醇,需用移液器将枪头在异丙醇吹吸两次后再缓慢吸取异丙醇。
2)向上一步的离心管中加入220 μL【20×(10+1)=220】Magbeads
注意:Magbeads加入前需涡旋振荡20秒使其充分混匀,异丙醇与Magbeads混合物使用前需涡旋振
荡20秒钟使其成为均一的溶液。
3)异丙醇与Magbeads混合物使用前需涡旋振荡10秒钟使其成为均一的溶液。
2.Magbeads严禁冰冻、离心。冰冻、离心可能会对Magbeads造成不可逆的损害。
3 .样品应避免反复冻融,否则会导致提取得到的DNA片段较小且提取得率低。
4.次使用前应按试剂瓶标签在Buffer GW1和Buffer GW2中加入无水乙醇并做好标记。
5 .如Buffer ML中出现沉淀,请在56℃水浴锅中重新溶解,摇匀后即可使用。
6.实验过程中,磁珠在溶液中的充分混匀对于提取的得率与纯度都有很大的影响。实 验过程中务必使磁珠与溶液充分混匀。不同厂家生产的恒温混匀仪震荡混匀效果有 一定差异,实验过程中请注意观察磁珠状态。如出现磁珠贴壁等未充分混匀的现象,
请用移液器吹吸混匀或调整震荡频率。
操作步骤(手动单管操作):
1.向1.5 mL的离心管中加入20 μL Proteinase K,之后加入200 μL的血液。
注意:1)冷冻抗凝血液需提前在室温(15-30℃)下放置,融化混匀。
2)如血液体积大于或小于200 μL,ProteinaseK、BufferML和异丙醇与磁珠混合物的用量需按比例调整。
2.向离心管中加入200 μL Buffer ML,涡旋振荡5秒钟使其充分混匀后,将离心管放于 56℃水浴锅中孵育15分钟,期间涡旋震荡混匀2次。
3.将离心管从水浴锅中取出,短暂离心后室温放置5分钟。加入彻底混匀的320 μL异丙醇 与Magbeads混合物,涡旋震荡混匀5秒钟后将离心管放于25℃、 1600 rpm的恒温混匀 仪上震荡混匀5分钟或将离心管连续颠倒混匀10分钟。
4.将离心管放于磁力架上静置1分钟,待Magbeads完全吸附于离心管侧壁后彻底弃去 溶液(保持离心管固定于磁力架上)
5 .将离心管从磁力架上取下,加入750 μL Buffer GW1(使用前请检查是否已加入无水 乙醇)后涡旋点震1分钟或涡旋振荡5秒钟后放于25℃、 1600 rpm的恒温混匀仪上震 荡混匀2分钟(震荡过程中确保Magbeads处于混匀状态)。之后将离心管放于磁力 架上静置1分钟, 待Magbeads完全吸附于离心管侧壁后轻轻颠倒磁力架将离心管盖 上的杂质洗落后彻底弃去溶液(保持离心管固定于磁力架上)。
6 .重复步骤5。
7 .将离心管从磁力架上取下,加入750 μL Buffer GW2(使用前请检查是否已加入无水 乙醇)后涡旋点震1分钟或涡旋振荡5秒钟后放于25℃、 1600 rpm的恒温混匀仪上震 荡混匀2分钟(震荡过程中确保Magbeads处于混匀状态)。之后将离心管放于磁力 架上静置1分钟, 待Magbeads完全吸附于离心管侧壁后轻轻颠倒磁力架将离心管盖 上的杂质洗落后彻底弃去溶液(保持离心管固定于磁力架上)。
8 .重复步骤7。
9 .保持离心管固定于磁力架上,用移液器进一步去除离心管管底和管盖上的溶液,之 后室温放置5-10分钟,使乙醇挥发干净。
注意:如果离心管侧壁上有液珠,可向离心管中加入750 μl无水乙醇。盖盖后颠倒离心管(保持离心管固
定于磁力架上),之后彻底弃去无水乙醇。
10 .将离心管从磁力架上取下,加入50-200 μL Buffer EB。涡旋震荡使磁珠完全悬浮于 洗脱液中后将其放于56℃、 1600 rpm的恒温混匀仪上震荡洗脱10分钟,或将离心 管放于56℃水浴锅中孵育10分钟,期间每隔3分钟涡旋震荡10秒钟。
11 .将离心管放于磁力架上静置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(手动96孔深孔板操作)
1.向2 mL 96孔深孔板(简称“深孔板” )中加入20 μL Proteinase K,之后加入200 μL血 液,并记录每孔中所加血液名称。
注意:1)可将ProteinaseK预先分装于8连管中,每管加入260 μL。之后,用8通道移液器将Proteinase K
分装于96孔深孔板中。
2)血液需加到96孔深孔板的底部,避免血液触碰到孔的上部。
2.向深孔板中用电动连续分液器或8通道移液器加入200 μL Buffer ML。
3.将深孔板固定于25℃、 1600 rpm的恒温混匀仪上震荡混匀2分钟,盖上硅胶盖后将 深孔板放于56℃水浴锅中孵育15分钟,之后再将深孔板固定于25℃、 1600 rpm的 恒温混匀仪上震荡混匀5分钟。
4.将深孔板从恒温混匀仪上取下,用电动连续分液器或8通道移液器向深孔板中加入 彻底混匀的320 μL Magbeads与异丙醇混合物。盖上硅胶盖后,立即将深孔板固定 于25℃、 1600 rpm的恒温混匀仪上震荡混匀5分钟。
5.将深孔板从恒温混匀仪上取下后放于96孔板磁力架上静置2分钟。用废液抽吸系统 或8通道移液器弃去溶液,期间避免枪头接触磁珠。
注意:用废液抽吸系统去除溶液时需将真空泵调节至较小的负压力值,使溶液以一个合适的速度被吸走,
速度过快会造成磁珠的丢失。
6.用手动连续分液器向深孔板中加入500 μL Buffer GW1(加入前检查是否已加入无水 乙醇),之后将深孔板固定于25℃、 1600 rpm的恒温混匀仪上震荡混匀3分钟。
7.将深孔板从恒温混匀仪上取下后放于96孔板磁力架上静置2分钟。用废液抽吸系统 或8通道移液器弃去溶液,期间避免枪头接触磁珠。
8.重复步骤6-7。
9.用手动连续分液器向深孔板中加入500 μL Buffer GW2(加入前检查是否已加入无水 乙醇),之后将深孔板固定于25℃、 1600 rpm的恒温混匀仪上震荡混匀3分钟。
10.将深孔板从恒温混匀仪上取下后放于96孔板磁力架上静置2分钟。用废液抽吸系统 或8通道移液器弃去溶液,期间避免枪头接触磁珠。
11.重复步骤9-10。
12.用手动连续分液器向96孔深孔板中加入500 μL无水乙醇,之后将深孔板固定于25℃、 1600 rpm的恒温混匀仪上震荡混匀1分钟。
13.将板从恒温混匀仪上取下后放于96孔板磁力架上静置2分钟。用废液抽吸系统或8 通道移液器弃去溶液,期间避免枪头接触磁珠。
14.保持深孔板固定于96孔板磁力架上,将深孔板倒置于干净的吸水纸上放置2分钟。 之后将深孔板从96孔板磁力架上取下放于100℃、 1600 rpm的恒温混匀仪上静置5 分钟。
15.用电动连续分液器或8通道移液器向深孔板加入100-200 μL Buffer EB,之后将深 孔板放于100℃(因该恒温混匀仪为悬空加热,洗脱液实际温度在50-60℃之间)、 1600 rpm的恒温混匀仪上震荡混匀10分钟。
16.将深孔板从恒温混匀仪上取下后放于96孔板磁力架上静置2分钟,用8通道移液器 溶液转移至96孔PCR板中盖盖后-20℃保存备用。
操作步骤(该产品与KingFisher Duo的匹配)
该产品与KingFisher Duo匹配后可从12份体积为200 μL的不同血液中提取基因组 DNA。
1 .按下表将样品与试剂加入相应位置
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2.启动软件BindIt,导入CoWin Magbind Blood DNA Duo-200程序。将加入样本与试剂 的DW 96深孔板与KF Duo洗脱条放入KingFisher Duo仪器中后执行程序CoWin Magbind Blood DNA Duo-200。
3.约12分钟后仪器暂停,取出DW 96深孔板,向A1-A12孔中加入320 μL彻底混匀的 Magbeads与异丙醇混合物。
4.将DW 96深孔板重新放入仪器中,继续运行程序。约26分钟后程序运行结束。
5.取出DW 96深孔板与KF Duo洗脱条,将洗脱条中的DNA溶液转移至1.5 mL离心管中, -20℃保存。
该产品与KingFisher Flex匹配后可从96份体积为200 μL的不同血液中提取血液基 因组DNA。
1 .按下表将样品与试剂加入相应板中:
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2 .启动软件BindIt,导入CoWin Magbind Blood DNA Flex-200程序。将加入试剂的DW 96 深孔板按顺序放入仪器中的相应位置后执行CoWin Magbind Blood DNA Flex-200程序。
3 .约14分钟后仪器暂停,取出样品板,向样品板中加入320 μL彻底混匀的Magbeads与异 丙醇混合物。
4 .将样品板重新放入仪器中,继续执行程序。约26分钟后程序执行完成。
5 .取出洗脱板,用膜封闭后-20℃保存。
Product content:
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Self provided instruments and reagents
1. Manual single tube extraction:
1) Constant temperature mixer
2) 2/15 mL magnetic rack
3) Isopropanol and anhydrous ethanol
2. Manual 96 hole deep hole plate extraction:
1) Constant temperature mixer
2) Waste liquid suction system
3) Manual continuous liquid separator
4) Electric continuous liquid separator
5) Manual continuous liquid separator pipette (25 mL)
6) 96 hole plate magnetic bracket
7) Isopropanol and anhydrous ethanol
3. Magnetic rod method magnetic bead automatic extraction system:
1) Automatic extraction system for magnetic beads using magnetic rod method
2) Isopropanol and anhydrous ethanol
Preparation and important precautions before the experiment:
1. Preparation of a mixture of isopropanol and Magheads (using the amount required to extract 10 samples as an example)
1) Add 3.3 mL [0.3 x (10+1)=3.3] of isopropanol to a centrifuge tube with an appropriate capacity (the total volume after adding isopropanol and Magheads is less than 2/3 of the centrifuge tube volume).
Attention: If using a pipette to add isopropanol, use a pipette to blow and aspirate the gun tip twice with isopropanol before slowly aspirating isopropanol.
2) Add 220 to the centrifuge tube in the previous step μ L [20 x (10+1)=220] Magheads
Attention: Before adding Magheads, vortex vibration is required for 20 seconds to fully mix them. Vortex vibration is required before using the mixture of isopropanol and Magheads Shake for 20 seconds to make it a homogeneous solution.
3) Before using the mixture of isopropanol and Magheads, vortex oscillation is required for 10 seconds to make it a homogeneous solution.
Magheads are strictly prohibited from freezing or centrifugation. Freezing and centrifugation may cause irreversible damage to Magheads.
3. Samples should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and lower extraction rates.
4. Before use, anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the label of the reagent bottle and labeled properly.
5. If sediment appears in the Buffer ML, please dissolve it again in a 56 ℃ water bath, shake well, and then use it.
6.During the experiment, the thorough mixing of magnetic beads in the solution has a significant impact on the extraction yield and purity. During the experiment, it is essential to thoroughly mix the magnetic beads with the solution. There are certain differences in the shaking and mixing effects of constant temperature mixers produced by different manufacturers. Please pay attention to the state of the magnetic beads during the experiment. If there is insufficient mixing such as magnetic beads sticking to the wall,
Please use a pipette to blow and suck, mix well, or adjust the oscillation frequency.
Operation steps (manual single tube operation):
1. Add 20 to a 1.5 mL centrifuge tube μ L Protein K, then add 200 μ L's blood.
Attention:
1) Frozen anticoagulant blood should be placed at room temperature (15-30 ℃) in advance, melted and mixed well.
2) If the blood volume is greater than or less than 200 μ L. The dosage of ProteinaseK, Bu ff erML, and the mixture of isopropanol and magnetic beads needs to be adjusted proportionally.
2. Add 200 to the centrifuge tube μ L Buffer ML, vortex for 5 seconds to mix thoroughly, then incubate the centrifuge tube in a 56 ℃ water bath for 15 minutes, during which vortex oscillates and mixes twice.
3. Remove the centrifuge tube from the water bath, centrifuge briefly, and let it sit at room temperature for 5 minutes. Add thoroughly mixed 320 μ Mix the mixture of L isopropanol and Magheads with vortex shaking for 5 seconds, then place the centrifuge tube on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and mix for 5 minutes, or invert the centrifuge tube continuously for 10 minutes.
4. Place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, completely discard the solution (keep the centrifuge tube fixed on the magnetic stand)
5. Remove the centrifuge tube from the magnetic frame and add 750 μ L Buffer GW1 (please check if anhydrous ethanol has been added before use), vortex point shake for 1 minute or vortex shake for 5 seconds, then place it on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and mix for 2 minutes (ensure that Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).
6. Repeat step 5.
7. Remove the centrifuge tube from the magnetic frame and add 750 μ L Buffer GW2 (please check if anhydrous ethanol has been added before use), vortex point shake for 1 minute or vortex shake for 5 seconds, and then place it on a constant temperature mixer at 25 ℃ and 1600 rpm to shake and mix for 2 minutes (ensure that Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).
8. Repeat step 7.
9. Keep the centrifuge tube fixed on the magnetic frame, use a pipette to further remove the solution from the bottom and cover of the centrifuge tube, and then leave it at room temperature for 5-10 minutes to allow the ethanol to evaporate completely.
Note: If there are liquid droplets on the sidewall of the centrifuge tube, 750 can be added to the centrifuge tube μ Anhydrous ethanol. Invert the centrifuge tube after covering (keep the centrifuge tube fixed) Set it on a magnetic frame, and then completely discard anhydrous ethanol.
10. Remove the centrifuge tube from the magnetic frame and add 50-200 μ L Buffer EB. Vortex oscillation causes the magnetic beads to completely suspend in the eluent and then place them on a constant temperature mixer at 56 ℃ and 1600 rpm for 10 minutes of shaking and elution, or incubate the centrifuge tube in a 56 ℃ water bath for 10 minutes, with vortex oscillation every 3 minutes for 10 seconds.
11. Place the centrifuge tube on a magnetic stand 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 at -20 ℃ for later use.
Operation steps (manual 96 hole deep hole plate operation)
1. Add 20 to 2 mL of 96 well deep well plate (referred to as "deep well plate") μ L Protein K, then add 200 μ L blood and record the name of the blood added in each well.
Note:
1) ProteinaseK can be pre packaged in 8 tubes, with 260 added to each tube μ L. Afterwards, use an 8-channel pipette to transfer Protein K
Divided into 96 hole deep hole plates.
2) Blood needs to be added to the bottom of the 96 well deep plate to avoid touching the upper part of the hole.
2. Add 200 to the deep orifice plate using an electric continuous separator or an 8-channel pipette μ L Buffer ML.
3. Fix the deep well plate on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and mix for 2 minutes. Cover with silicone gel and incubate the deep well plate in a 56 ℃ water bath for 15 minutes. Then, fix the deep well plate on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and mix for 5 minutes.
4. Remove the deep well plate from the constant temperature mixer, and use an electric continuous separator or 8-channel pipette to add thoroughly mixed 320 to the deep well plate μ A mixture of L Magheads and isopropanol. After covering with silicone, immediately fix the deep well plate on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and mix for 5 minutes.
5. Remove the deep well plate from the constant temperature mixer and place it on the 96 well plate magnetic stand for 2 minutes. Discard the solution using a waste liquid suction system or an 8-channel pipette, while avoiding contact between the gun tip and the magnetic beads.
Attention: When using a waste liquid suction system to remove the solution, the vacuum pump needs to be adjusted to a smaller negative pressure value to allow the solution to be sucked away at an appropriate speed,
Excessive speed can cause the loss of magnetic beads.
6. Use a manual continuous separator to add 500 to the deep orifice plate μ L Buffer GW1 (check if anhydrous ethanol has been added before adding), then fix the deep well plate on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and mix for 3 minutes.
7. Remove the deep well plate from the constant temperature mixer and place it on the 96 well plate magnetic stand for 2 minutes. Discard the solution using a waste liquid suction system or an 8-channel pipette, while avoiding contact between the gun tip and the magnetic beads.
8. Repeat steps 6-7.
9. Use a manual continuous liquid separator to add 500 to the deep orifice plate μ L Buffer GW2 (check if anhydrous ethanol has been added before adding), then fix the deep well plate on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and mix for 3 minutes.
10. Remove the deep well plate from the constant temperature mixer and place it on the 96 well plate magnetic stand for 2 minutes. Discard the solution using a waste liquid suction system or an 8-channel pipette, while avoiding contact between the gun tip and the magnetic beads.
11. Repeat steps 9-10.
12. Use a manual continuous liquid separator to add 500 to the 96 well deep well plate μ L anhydrous ethanol, then fix the deep well plate on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and mix for 1 minute.
13. Remove the plate from the constant temperature mixer and place it on a 96 well plate magnetic stand for 2 minutes. Discard the solution using a waste liquid suction system or an 8-channel pipette, while avoiding contact between the gun tip and the magnetic beads.
14. Keep the deep hole plate fixed on the 96 hole plate magnetic bracket, invert the deep hole plate on clean absorbent paper and let it stand for 2 minutes. Afterwards, take the deep well plate from the 96 well plate magnetic holder and place it on a constant temperature mixer at 100 ℃ and 1600 rpm, and let it stand for 5 minutes.
15. Use an electric continuous separator or 8-channel pipette to add 100-200 to the deep orifice plate μ L Buffer EB, then place the deep well plate on a 100 ℃ (as the constant temperature mixer is heated in the air, the actual temperature of the eluent is between 50-60 ℃) and a 1600 rpm constant temperature mixer, shake and mix for 10 minutes.
16. Remove the deep well plate from the constant temperature mixer and place it on a 96 well plate magnetic stand for 2 minutes. Use an 8-channel pipette to transfer the solution to the 96 well PCR plate and cover it. Store at -20 ℃ for later use.
Operation steps (matching the product with KingFisher Duo)
This product can be paired with KingFisher Duo from 12 parts to a volume of 200 μ Extract genomic DNA from different blood samples of L.
1. Add the samples and reagents to the corresponding positions according to the following table:
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2. Start the software BindIt and import the CoWin Magbind Blood DNA Duo-200 program. Place the DW 96 deep well plate and KF Duo elution strip with added samples and reagents into the KingFisher Duo instrument and execute the CoWin Magbind Blood DNA Duo-200 program.
3. After about 12 minutes, the instrument will pause. Remove the DW 96 deep well plate and add 320 to the A1-A12 holes μ A thoroughly mixed mixture of Magheads and isopropanol.
4. Insert the DW 96 deep well plate back into the instrument and continue running the program. The program will finish running in about 26 minutes.
5. Take out the DW 96 deep well plate and KF Duo elution strip, transfer the DNA solution from the elution strip to a 1.5 mL centrifuge tube, and store at -20 ℃.
This product can be paired with KingFisher Flex from 96 parts to a volume of 200 μ Extract genomic DNA from different blood samples of L.
1. Add the sample and reagent to the corresponding plate according to the table below:
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2. Start the software BindIt and import the CoWin Magbind Blood DNA Flex-200 program. Place the DW 96 deep well plate with added reagents in the corresponding positions in the instrument in order and execute the CoWin Magbind Blood DNA Flex-200 program.
3. After about 14 minutes, the instrument will pause, remove the sample plate, and add 320 to the sample plate μ A thoroughly mixed mixture of Magheads and isopropanol.
4. Place the sample board back into the instrument and continue with the program. The program execution is completed in approximately 26 minutes.
5. Remove the elution plate, seal it with a membrane, and store it at -20 ℃.