基本描述

英文名称

Blood Genomic DNA Midi Kit (1-5 mL)

储存温度

2-8°C储存

运输条件

冰袋运输

产品介绍

产品内容

B669892	Component	50 T	Storage
B669892A	Buffer RCL	3×260 mL	2-8℃
B669892B	Buffer GR	25 mL	RT
B669892C	Buffer GL	25 mL	RT
B669892D	Buffer GW1 (concentrate)	13 mL	RT
B669892E	Buffer GW2 (concentrate)	15 mL	RT
B669892F	Buffer GE	15 mL	RT
B669892G	Proteinase K	50 mg	RT
B669892H	Proteinase K Storage Buffer	5 mL	RT
B669892I	Spin Columns DL with Collection Tubes	50 sets	RT

产品简介

本试剂盒适用于从新鲜或冷冻全血（用柠檬酸盐、EDTA或肝素等抗凝剂处理过的血液样品）、血浆、血清、血沉棕黄层、骨髓、无细胞体液等样本中提取总DNA，包括基因组DNA，线粒体DNA及病毒DNA。本品可以处理1-5 ml的全血，可纯化获得大小为100 bp到50 kb的DNA，纯化的DNA产量高、质量好，最大限度去除蛋白、色素、脂类及其他抑制性杂质，可直接用于PCR、荧光定量PCR、酶切和Southern Blot等实验。

自备试剂：无水乙醇。

实验前准备及重要注意事项

1. 向Proteinase K中加入5ml Proteinase K Storage Buffer使其溶解，-20℃保存。配制好的Proteinase K勿长时间室温放置，避免反复冻融，以免影响其活性。

2. 样品应避免反复冻融，否则会导致提取的DNA片段较小且提取量下降。

3. 本试剂盒最多可以提取1-5ml全血样品，如需提取大量血液样本请选用血液基因组非柱式提取试剂盒。

4. 第一次使用前应按试剂瓶标签说明先在Buffer GW1和Buffer GW2中加入无水乙醇。

5. 使用前请检查Buffer GL是否出现结晶或者沉淀，如有结晶或者沉淀，请置于56℃水浴重新溶解。

6. 如下游实验对RNA污染较敏感，可加入4μl DNase Free的RNase A（100 mg/ml）， RNase A本试剂盒并未提供，如需要可单独向本公司订购。

7. 试剂盒中的Buffer RCL浑浊后不能继续使用。

操作步骤

1．向离心管（自备）中加入1-5 ml血液样品，加入3倍体积的Buffer RCL轻轻涡旋或颠倒混匀。

2．3000 rpm（～900 x g）离心10分钟，小心吸弃上清。

3．向沉淀中加入400 μl Buffer GR，重悬沉淀。

注意：如果下游试验对RNA敏感，可加入4 μl RNase A（100 mg/ml）溶液，震荡15秒，室温放置5分钟。

4．1-2 ml血液样本提取时,向以上溶液中加入40 μl Proteinase K，混匀；2-5ml血液样本提取时,向以上溶液中加入100 μl Proteinase K，混匀。

5．加入400 μl Buffer GL，颠倒混匀15次，剧烈涡旋震荡至少1分钟。

注意：不要直接将Proteinase K直接加入到Buffer GL中。

6．70℃孵育10分钟，其间颠倒混匀数次。

注意：1）如溶液未彻底清亮，补加适量Proteinase K，孵育。延长孵育时间至溶液完全清亮为止。

2）孵育10分钟DNA的产量已经达到最大，继续延长孵育时间对DNA产量和纯度没有影响。

7. 加入400 μl无水乙醇，颠倒混匀10次。短暂离心，使管壁和管盖上液体集中到管底。

8. 将上步所得到的溶液全部加入到已装入收集管的吸附柱（Spin Columns DL）中，若一次不能加完溶液，可分多次转入。12,000 rpm（~13,400×g）离心1分钟，倒掉收集管中的废液，将吸附柱重新放回收集管中。

9. 向吸附柱中加入500 μl Buffer GW1（使用前检查是否加入无水乙醇），12,000 rpm离心1分钟，倒掉收集管中的废液，将吸附柱重新放回收集管中。

注意：如果提取样品是小鼠或猴子等血红素难以除去的种属的血液基因组，建议重复步骤9。

10. 向吸附柱中加入500 μl Buffer GW2（使用前检查是否加入无水乙醇），12,000 rpm离心1分钟，倒掉收集管中的废液，将吸附柱重新放回收集管中。

注意：如需进一步提高DNA纯度，可重复步骤10。

11.12,000 rpm离心2分钟，倒掉收集管中的废液。将吸附柱置于室温数分钟，以彻底晾干。

注意：这一步的目的是将吸附柱中残余的乙醇去除，乙醇的残留会影响后续的酶促反应（酶切、PCR 等）

12.将吸附柱置于一个新的离心管中，向吸附柱中间部位悬空加入50-200 μl Buffer GE 或灭菌水，室温下放置2-5分钟，12,000 rpm离心1分钟，收集DNA溶液，-20℃保存DNA。

注意：1）如果下游实验对pH值或EDTA敏感，可以用灭菌水洗脱。洗脱液的pH值对洗脱效率有很大影响，若用水做洗脱液应保证其pH值在7.0-8.5（可以用NaOH将水的pH值调到此范围），pH值低于7.0时洗脱效率不高。

2） 离心之前室温孵育5分钟可以增加产量。

3） 用另外的50-200 μl Buffer GE或灭菌水再次洗脱可以增加产量。

4） 如果要提高DNA的终浓度，可以将步骤12所得的DNA洗脱液重新加至吸附膜上，12,000 rpm离心

1min；若洗脱体积小于200 μl，可以增加DNA的终浓度，但可能会减少总产量。如果DNA的量小于1 μg，推荐用50 μl Buffer GE或灭菌水洗脱。

5） 因为保存在水中的DNA会受到酸性水解作用的影响，如需长期保存，推荐用Buffer GE洗脱并于-20℃保存。

Products

B669892	Component	50 T	Storage
B669892A	Buffer RCL	3×260 mL	2-8℃
B669892B	Buffer GR	25 mL	RT
B669892C	Buffer GL	25 mL	RT
B669892D	Buffer GW1 (concentrate)	13 mL	RT
B669892E	Buffer GW2 (concentrate)	15 mL	RT
B669892F	Buffer GE	15 mL	RT
B669892G	Proteinase K	50 mg	RT
B669892H	Proteinase K Storage Buffer	5 mL	RT
B669892I	Spin Columns DL with Collection Tubes	50 sets	RT

Products

This kit is suitable for the extraction of total DNA, including genomic DNA, mitochondrial DNA and viral DNA, from fresh or frozen whole blood (blood samplestreated with anticoagulants such as citrate, EDTA or heparin), plasma, serum, haematocrit brown and yellow layers, bone marrow, cell-free body fluids, etc. Theproduct can process 1-5 ml of whole blood, and can be purified to obtain sizes rangingfrom 100bp to 50kb. The purified DNA is of high yield and good quality, with maximumremoval of proteins, pigments, lipids and other inhibitory impurities, and can bedirectly used in PCR, fluorescence quantitative PCR, enzyme digestion and SouthernBlot.

Self-contained reagent: anhydrous ethanol.

Pre-experiment Preparation and Important Notes

1. Add 5ml Proteinase K Storage Buffer to Proteinase K to dissolve it, and storeit at -20℃. Do not leave the prepared Proteinase K at room temperature for a longtime, and avoid repeated freezing and thawing to avoid affecting its activity.

2. Repeated freezing and thawing of the sample should be avoided, as this may resultin smaller DNA fragments and a decrease in the amount of extracted DNA. 3.This kit can extract up to 1-5 ml of whole blood samples, if you need to extracta large number of blood samples, please use the blood genome non-column extractionkit.

4. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to theinstructions on the label of the reagent bottle before first use.

5. Please check Buffer GL for crystallization or precipitation before use, if thereis any crystallization or precipitation, please put it in 56℃water bath to re-dissolve.

6. If the downstream experiments are sensitive to RNA contamination, 4μl of DNaseFree RNase A (100mg/ml) can be added, RNase A is not provided in the kit, and canbe ordered separately from our company if needed.

7. The Buffer RCL in the kit cannot be used further after turbidity.

procedure

1. Add 1-5 ml of blood sample to a centrifuge tube (supplied) and add 3 times thevolume of Buffer RCL and gently vortex or invert to mix.

2. Centrifuge at 3000 rpm (~900 x g) for 10 minutes and carefully aspirate thesupernatant.

3. Add 400 μl Buffer GR to the precipitate and resuspend the precipitate. Note: If the downstream assay is sensitive to RNA, add 4 μl of RNase A (100 mg/ml)solution, shake for 15 seconds, and leave at room temperature for 5 minutes.

4. For 1-2 ml blood sample extraction, add 40μl Proteinase K to the above solutionand mix well; for 2-5 ml blood sample extraction, add 100μl Proteinase K to theabove solution and mix well.

5. Add 400 μl of Buffer GL, mix upside down 15 times, and vigorously vortex andshake for at least 1 minute. Note: Do not add Proteinase K directly to Buffer GL.

6. Incubate at 70°C for 10 minutes, during which time mixing was inverted severaltimes.

Note: 1) If the solution is not completely clear, add appropriate amount of Proteinase K and incubate. Extend the incubation time until the solution is completely clear. 2) The yield of DNA has been maximized by 10 minutes of incubation, and continuedprolongation of the incubation time has no effect on DNA yield or purity.

7. Add 400 μl of anhydrous ethanol and mix upside down 10 times. Centrifuge brieflyto concentrate the liquid on the walls and cap to the bottom of the tube.

8. Add all of the solution obtained in the previous step to the Spin Columns DL inthe collection tube. If the solution cannot be added all at once, transfer it severaltimes. centrifuge at 12,000 rpm (~13,400 x g) for 1 minute, pour off the waste liquidfrom the collection tube, and put the column back into the collection tube.

9. Add 500 μl of Buffer GW1 to the adsorption column (check that anhydrous ethanolis added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquidin the collection tube, and put the adsorption column back into the collection tube.

Note: It is recommended that step 9 be repeated if the sample being extracted isthe blood genome of a species such as mice or monkeys from which hemoglobin isdifficult to remove.

10. Add 500 μl Buffer GW2 to the adsorption column (check that anhydrous ethanolis added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquidin the collection tube, and put the adsorption column back into the collection tube.Note: Step 10 can be repeated if further DNA purity is required.

11. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in thecollection tube. Leave the adsorption column at room temperature for several minutesto dry thoroughly. Note: The purpose of this step is to remove residual ethanol from the adsorptioncolumn, which can interfere with subsequent enzymatic reactions (digestion, PCR,etc.)

12. Place the adsorption column in a new centrifuge tube, add 50-200 μl of BufferGE or sterilized water to the middle of the adsorption column overhanging the column,leave it at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute,collect the DNA solution, and store the DNA at -20℃.

Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on theelution efficiency, if water is used as the eluent should ensure that its pH is7.0-8.5 (you can use NaOH to adjust the pH of the water to this range), and the elutionefficiency is not high when the pH is lower than 7.0.

2) Incubation at room temperature for 5 minutes prior to centrifugation increasesyield.

3) Re-elution with an additional 50-200 μl Buffer GE or sterilized water can increase the yield.

4) If the final concentration of DNA is to be increased, the DNA eluate obtainedin step 12 can be re-spiked onto the adsorbent membrane and centrifuged at 12,000rpm. 1min; if the elution volume is less than 200μl, the final concentration of DNA canbe increased, but the total yield may be reduced. If the amount of DNA is less than1 μg, elution with 50 μl Buffer GE or sterilized water is recommended.

5) Because DNA preserved in water is subject to acidic hydrolysis, for long-termstorage, it is recommended that it be eluted with Buffer GE and stored at -20℃.

安全和危险性（GHS）

SDS英文版

质量标准

质量标准

b669892_blood genomic dna midi kit(1-5ml).pdf

质检证书（CoA,COO,BSE/TSE 和分析图谱)

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

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血液基因组柱式中量提取试剂盒 (1-5 mL)

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