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NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells
NP-40 提取分选哺乳动物细胞核酸   

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参见作者原研究论文

本实验方案简略版
Journal of Virology
Jun 2014

Abstract

This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear and perinuclear associated components in the nuclear fraction. This protocol has been modified from Sambrook and Russell (2001) in order to downscale the amount of cells needed. To determine the efficiency of fractionation, we recommend using qPCR to compare the subcellular compartments that have been purified with equivalent amount of control whole cell extracts.

Keywords: Cell fractionation (细胞分级分离), Cytoplasmic and nuclear extractions (细胞质和细胞核提取), Nucleic acid purification (核酸纯化), Quantitative PCR (定量PCR)

Background

To fully obtain an understanding of cellular processes, fractionation of nuclear and cytoplasmic compartments are needed. There are many protocols and even some commercial kits available to help separate the two compartments. However, most require high centrifugation speeds and there is a high discrepancy in the yield and even the methods to verify the amount of contamination in the final products. Our protocol utilizes a small benchtop centrifuge at low speeds to obtain highly pure extractions for the cytoplasmic and a combined nuclear/perinuclear associated compartments as well as the data analysis to verify the percentage of contamination. To date, the cells lines that have been tested are 293 T, HeLa and GHOST cell lines. (Galvis, 2014; Galvis et al., 2014).

Materials and Reagents

  1. BD 21 G needles (Fisher Scientific, catalog number: 14-823-55)
    Manufacturer: BD, catalog number: 305274 .
  2. BD tuberculin syringes (Fisher Scientific, catalog number: 14-823-2F)
    Manufacturer: BD, catalog number: 309602 . This product has been discontinued.
  3. TipOne 10 µl pipet tips (USA Scientific, catalog number: 1111-3200 )
  4. TipOne 1-200 µl natural pipet tips (USA Scientific, catalog number: 1111-1800 )
  5. TipOne 1,000 µl natural pipet tips (USA Scientific, catalog number: 1111-2020 )
  6. Costar 6-well cell culture plates (Fisher Scientific, catalog number: 07-200-80)
    Manufacturer: Corning, catalog number: 3506 .
  7. 1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 14-666-318 )
  8. Iscove’s modification of DMEM (Mediatech, catalog number: 10-016-CV )
  9. Tween 40 (CHEM-IMPEX INTERNATIONAL, catalog number: 01513 )
  10. 1x phosphate buffered saline (PBS) (MP Biomedicals, catalog number: 091860454 )
  11. 1x trypsin-EDTA (Mediatech, catalog number: 25-051-Cl )
  12. 0.5 M ethylenediaminetetraacetic acid (EDTA) pH 8.0 (Fisher Scientific, catalog number: BP2482-500 )
  13. 10% sodium dodecyl sulfate (SDS) (Mediatech, catalog number: 46-040-Cl )
  14. Proteinase K (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0491 )
  15. Ribonuclease A, DNase free (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0531 )
  16. Potassium acetate (Fisher Scientific, catalog number: P171-500 )
  17. Isopropanol (Fisher Scientific, catalog number: A416-500 )
  18. Ethanol 70% (Fisher Scientific, catalog number: BP82011 )
  19. Sodium hydroxide (NaOH) (Fisher Scientific, catalog: S318-500 )
  20. SYBR Green PCR Master mix (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4309155 )
  21. Magnesium chloride (MgCl2) (Fisher Scientific, catalog number: BP214-500 )
  22. Sucrose (Fisher Scientific, catalog number: S3-500 )
  23. Tris-HCl buffer pH 7.4 (Lonza, catalog number: 51237 )
  24. Potassium chloride (KCl) (Fisher Scientific, catalog number: P330-500 )
  25. NP-40 10% solution (Thermo Fisher Scientific, catalog number: 28324 )
  26. 1 M MgCl2 (see Recipes)
  27. 2.5 M sucrose (see Recipes)
  28. TMK (see Recipes)
  29. TMK + 1% NP-40 (see Recipes)
  30. S1 buffer (see Recipes)
  31. S2 buffer (see Recipes)
  32. Cell lysis buffer (see Recipes)

Equipment

  1. Ventilated microcentrifuge (Fisher Scientific, model: accuSpinTM Micro 17R, catalog number: 13-100-676 )
  2. Gilson PIPETMAN Classic pipets (Gilson, model: P1000, catalog number: F123602 )
  3. Gilson PIPETMAN Classic pipets (Gilson, model: P20, catalog number: F123600 )
  4. Gilson PIPETMAN Classic pipets (Gilson, model: P200, catalog number: F123601 )
  5. Gilson PIPETMAN Classic pipets (Gilson, model: P2, catalog number: F144801 )
  6. Water bath (Fisher Scientific, model: FS-14 )
  7. Vortexer (Fisher Scientific, catalog number: 02-216-108 )
  8. ABI Prism 7900HT sequence detection system (PE-Applied Biosystems) (Thermo Fisher Scientific, Applied BiosystemsTM, model: ABI PRISMTM 7900HT )

Procedure

  1. NP-40 fractionation
    1. Wash plate 2 x with 2 ml PBS per well. Plate should have 5-10 x 106 cells at 90-95% confluence.
    2. Trypsinize cells (500 μl/well) and add 1 ml/well of media to neutralize. Combine the 6 wells of each plate. Can use a single 10 cm dish at the same confluence of 90-95% with 3-6 x107 cells.
    3. Spin cells at 4 °C, 100 x g for 5 min.
    4. Resuspend cells with 1 ml PBS.
    5. Pellet cells at 100 x g for 4 min at 4 °C.
    6. Resuspend cells in 200 μl TMK.
      Note: For qPCR analysis of whole cell extraction, you can take out 50 μl and extract the DNA before proceeding to Procedure B.
    7. Add 150 μl of TMK +1% NP-40.
    8. Incubate on ice for 5 min with occasional gentle vortexing.
    9. Pellet at 228 x g for 5 min at 4 °C.
    10. Remove the supernatant without disturbing the pellet–This is the Cytoplasmic fraction.
    11. Suspend the stripped nuclear pellet in 500 ml of S1 buffer.
    12. Layer the S1 suspended pellets over 500 ml of S2 buffer.
    13. Centrifuge at 1,430 x g for 5 min at 4 °C.
    14. Aspirate supernatant off.
    15. Resuspend the nuclei in 300 μl PBS.
      Note: At this point, all samples can be stored at 4 °C overnight before proceeding to Procedure B.

  2. DNA extractions
    1. Add 600 μl ice-cold cell lysis buffer to the cell extracts. Expect SDS precipitation with most extractions; it will not affect the end result. Vortex thoroughly for 30 sec.
    2. Add 3 μl of Proteinase K solution (20 mg/ml). Incubate DNA sample at 55 °C for 3-16 h. The time is dependent on how much SDS precipitation is noticed on the samples and cell line used. A rule of thumb is the more SDS precipitation and larger cells tend to require more incubation time.
    3. Shear DNA by drawing repeatedly (15 x) through a 21 G needle, avoiding excessive foaming and loss by aerosolization.
    4. Allow cooling to RT, then add 3 μl of 4 mg/ml DNAse-free Ribonuclease A. Digest for 15-60 min at 37 °C.
      Note: At this point, all samples can be stored at 4 °C overnight before proceeding to step B5.
    5. Cool to RT. Add 200 μl 5 M potassium acetate; vortex for 20 sec.
    6. Pellet precipitated protein/SDS by centrifugation at 4 °C for 3 min at maximum RPM.
    7. Transfer solution to a fresh tube with 600 μl isopropanol. Mix by inversion. Spin at maximum RPM for 3 min at RT.
    8. Remove supernatant and wash DNA with 600 μl 70% EtOH. Spin for 3 min at maximum RPM to sediment the pellet. Aspirate the ethanol. Repeat with a 1-min spin and aspirate residual EtOH.
    9. Air-dry for 15 min.
    10. Redissolve the pellet in 100 μl 8 mM NaOH or 10 mM Tris. This may be slow–an incubation at 37 °C for 15 min is recommended.
    11. The DNA may now be stored at -20 °C without incident.

  3. Assessing efficiency of the NP-40 fractionation
    To assess the accuracy of the nucleo-cytoplasmic separation, qPCR can be utilized using nuclear, mitochondrial and whole cell extract samples such as example below.
    1. DNA samples were diluted 1 to 10 in dH2O and incubated at 98 °C for 20 min followed by immediate cooling on ice.
    2. Each PCR reaction contained 7.5 μl SYBR Green PCR Master mix (Fermentas), 2 μl of template, and 1 μM of each primer (Table 1) in a 15 μl reaction volume.

      Table 1. Primers for DNA amplification


    3. QPCR was performed in triplicate for each sample using an ABI Prism 7900HT sequence detection system (PE-Applied Biosystems) for amplification and detection. PCR conditions were as follows:

Data analysis

  1. The accuracy of the NP-40 nucleo-cytoplasmic fraction can be verified by comparing the amount of mitochondrial DNA copies detected in the nuclear fraction and the amount of β-globin DNA copies in the cytoplasmic fraction with the respective copies found in the total cell lysate. The data is analyzed by delta method which requires an average threshold cycle and standard deviation. Hence,
    Ct = threshold cycle, a = nuclear or cytoplasmic sample, b = whole cell lysate sample:



  2. The yield recovered is compared to cytoplasmic contamination in nuclear fractions. Figure 1 adapted from Galvis (2014) is an example of the data analysis graphs that can be composed with the obtained data. The ratio of mitochondrial DNA copies in the nuclear fractions to mitochondrial DNA copies in the whole cell lysate. The example demonstrates a successful fractionation. The nuclear fraction has a 1:1 ratio of nuclear DNA with the whole cell extract; while at the same time there is minimal mitochondrial DNA in comparison to the whole cell extract. The cytoplasmic fraction contains minimal nuclear DNA in comparison to the whole cell extract while it contains an almost 1:1 ratio of mitochondrial DNA with the whole cell extract.
  3. Note that the recommended inclusion data is that all triplicates must be within one cycle apart from each other and all obtained cycles must be smaller than the negative control that need to be included in the qPCR runs.


    Figure 1. Sample data analysis of fractionation protocol. This is a ratio of mitochondrial and β-globin DNA in nuclear and cytoplasmic fractions vs. total cell lysates. GHOST-R5X4 cells were transfected either with DBR1 shRNA pHyper-D4 (D4) or DBR1 shRNA triple mismatch, pHyper-M4 (M4). Two to twenty-four hours later, the cells were either fractionated into nuclear and cytoplasmic fractions or lysed to prepare whole cell extracts. DNA was isolated for qPCR to evaluate the success of fractionation. A. β-Globin primers were utilized to assay the exclusion of nuclear contamination in the cytoplasmic fraction in contrast to whole cell extracts. The graph illustrates the ratio of β-globin copies in the cytoplasmic fractions to β-globin copies in the whole cell lysates. B. β-Globin primers were utilized to assay concentration of nuclear fractions compared to whole cell extracts. The graph illustrates the ratio of β-globin copies in the nuclear fractions to β-globin copies in the whole cell lysate. C. Mitochondrial primers were used to assay concentration of cytoplasmic fraction compared to whole cell extracts. The graph illustrates the ratio of mitochondrial DNA copies in the nuclear fractions to β-globin copies in the whole cell lysates. D. Mitochondrial primers were utilized to assay the exclusion of mitochondrial contamination in the nuclear extractions.

Notes

  1. The data presented in Procedure C were the unpublished controls for experiments denoted in Galvis et al., 2014. Debranching enzyme 1 (DBR1) in our experiments has been implicated in reverse transcription of the HIV-1 genome.
  2. In Procedure A step 12, it is very important not to disturb the pellet and hence it is better to leave some of the supernatant behind. The purpose of the subsequent steps is to remove the remaining mitochondrial DNA.
  3. In our experience, the resuspension of the DNA with 8 mM NaOH has given us better overall recovery yield for downstream applications in comparison to 10 mM Tris. However, if the experiments desired only need for qPCR purposes only and not sequencing then 10 mM Tris is sufficient for resuspension of the final pellet.

Recipes

  1. 1 M MgCl2 (500 ml)
    Add 101.65 g MgCl2·6H2O into 500 ml ddH2O
    Store at room temperature
  2. 2.5 M sucrose
    8.55 g of sucrose
    Up to 10 ml with ddH2O
    Need to heat to 37 °C in order to have sucrose fully dissolve
    Store at room temperature
  3. TMK (10 ml; store in 4 °C)


  4. TMK + 1% NP-40 (2 ml)
    1.8 ml TMK
    200 μl 10% NP-40
    Store in 4 °C
  5. S1 buffer
    0.25 M sucrose
    10 mM MgCl2


  6. S2 buffer
    0.35 M sucrose
    0.5 mM MgCl2
    Store at 4 °C


  7. Cell lysis buffer
    10 mM Tris-HCl, pH 7.4
    0.1% SDS
    1 mM EDTA
    Store at 4 °C

Acknowledgments

This work was supported in part by grant ID07-I-124 from the California HIV-AIDS Research Program to David Camerini. Support of the UCI Center for Virus Research, UCI Cancer Research Institute and the Chao Family Comprehensive Cancer Center are acknowledged. Alvaro Galvis was supported by the UCI Medical Scientist Training Program, grant T32-GM08620. This protocol is adapted and modified from the original in Sambrook and Russell, 2001.

References

  1. Galvis, A. E. (2014). An RNA lariat intermediate in HIV-1 cDNA synthesis. ProQuest 3615193.
  2. Galvis, A. E., Fisher, H. E., Nitta, T., Fan, H. and Camerini, D. (2014). Impairment of HIV-1 cDNA synthesis by DBR1 knockdown. J Virol 88(12): 7054-7069.
  3. Sambrook, J. and Russell, D. W. (2001). Molecular cloning: a laboratory manual. CSHL Press.

简介

该技术允许在哺乳动物细胞中利用NP-40洗涤剂进行高效,高度纯化的细胞质和核相关的分室分离。 在分离过程中核膜不受干扰,从而使核部分中的所有核和核周相关成分留下。 该协议已经从Sambrook和Russell(2001)修改,以便缩减所需的细胞数量。 为了确定分馏的效率,我们建议使用qPCR来比较已经用等量的对照全细胞提取物纯化的亚细胞室。
【背景】为了充分获得对细胞过程的理解,需要分离核和细胞质隔室。 有许多协议,甚至一些商业套件可用于帮助分离两个隔间。 然而,大多数需要高离心速度,产量差异甚至验证最终产品中的污染物量的方法也是很高的。 我们的协议在低速下使用小型台式离心机,以获得高纯度的细胞质提取物和核/核周组合相关隔室,以及数据分析,以验证污染物的百分比。 迄今为止,已经测试的细胞系是293T,HeLa和GHOST细胞系。 (Galvis,2014; Galvis等,,2014)。

关键字:细胞分级分离, 细胞质和细胞核提取, 核酸纯化, 定量PCR

材料和试剂

  1. BD 21 G针(Fisher Scientific,目录号:14-823-55)
    制造商:BD,目录号:305274。
  2. BD结核菌注射器(Fisher Scientific,目录号:14-823-2F)
    制造商:BD,目录号:309602.该产品已停产。
  3. TipOne 10μl移液管提示(USA Scientific,目录号:1111-3200)
  4. TipOne 1-200μl天然吸头(USA Scientific,目录号:1111-1800)
  5. TipOne 1000μl天然吸头(USA Scientific,目录号:1111-2020)
  6. Costar 6孔细胞培养板(Fisher Scientific,目录号:07-200-80)
    制造商:Corning,目录号:3506。
  7. 1.5ml微量离心管(Fisher Scientific,目录号:14-666-318)
  8. Iscove修改DMEM(Mediatech,目录号:10-016-CV)
  9. 吐温40(CHEM-IMPEX INTERNATIONAL,目录号:01513)
  10. 1x磷酸缓冲盐水(PBS)(MP Biomedicals,目录号:091860454)
  11. 1x胰蛋白酶-EDTA(Mediatech,目录号:25-051-Cl)
  12. 0.5M乙二胺四乙酸(EDTA)pH 8.0(Fisher Scientific,目录号:BP2482-500)
  13. 10%十二烷基硫酸钠(SDS)(Mediatech,目录号:46-040-Cl)
  14. 蛋白酶K(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EO0491)
  15. 核糖核酸酶A,无DNA酶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EN0531)
  16. 醋酸钾(Fisher Scientific,目录号:P171-500)
  17. 异丙醇(Fisher Scientific,目录号:A416-500)
  18. 乙醇70%(Fisher Scientific,目录号:BP82011)
  19. 氢氧化钠(NaOH)(Fisher Scientific,目录:S318-500)
  20. SYBR Green PCR Master混合物(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4309155)
  21. 氯化镁(MgCl 2)(Fisher Scientific,目录号:BP214-500)
  22. 蔗糖(Fisher Scientific,目录号:S3-500)
  23. Tris-HCl缓冲液pH 7.4(Lonza,目录号:51237)
  24. 氯化钾(KCl)(Fisher Scientific,目录号:P330-500)
  25. NP-40 10%溶液(Thermo Fisher Scientific,目录号:28324)
  26. 1 M MgCl 2(见配方)
  27. 2.5 M蔗糖(见食谱)
  28. TMK(见配方)
  29. TMK + 1%NP-40(见配方)
  30. S1缓冲(见配方)
  31. S2缓冲区(见配方)
  32. 细胞裂解缓冲液(参见食谱)

设备

  1. 通风微量离心机(Fisher Scientific,型号:accuSpin TM Micro 17R,目录号:13-100-676)
  2. Gilson PIPETMAN经典移液器(Gilson,型号:P1000,目录号:F123602)
  3. Gilson PIPETMAN经典移液器(Gilson,型号:P20,目录号:F123600)
  4. Gilson PIPETMAN经典移液器(Gilson,型号:P200,目录号:F123601)
  5. Gilson PIPETMAN经典移液器(Gilson,型号:P2,目录号:F144801)
  6. 水浴(Fisher Scientific,型号:FS-14)
  7. Vortexer(Fisher Scientific,目录号:02-216-108)
  8. ABI Prism 7900HT序列检测系统(PE-Applied Biosystems)(Thermo Fisher Scientific,Applied Biosystems TM,型号:ABI PRISM TM> 7900HT)

程序

  1. NP-40分馏
    1. 每孔用2ml PBS洗涤2次。平板应该在90-95%汇合处具有5-10×10 6个细胞。
    2. 胰蛋白酶消化细胞(500μl/孔)并加入1ml /孔的培养基中和。结合每个板的6个孔。可以使用单一的10厘米盘,同样的90-95%的汇合点与3-6×10 7个细胞。
    3. 在4℃,100×g下旋转细胞5分钟。
    4. 用1ml PBS重悬细胞。
    5. 颗粒细胞在100℃下在4℃下4分钟。
    6. 将细胞重悬于200μlTMK。
      注意:对于全细胞提取的qPCR分析,您可以在进行步骤B之前取出50μl并提取DNA。
    7. 加入150μlTMK + 1%NP-40。
    8. 在冰上孵育5分钟,偶尔轻轻摇动。
    9. 颗粒在228℃下在4℃下5分钟。
    10. 去除上清液而不干扰沉淀物 - 这是细胞质级分。
    11. 将剥离的核丸悬浮在500ml S1缓冲液中
    12. 将S1悬浮颗粒层叠在500ml S2缓冲液上
    13. 在4℃下以1,430×g离心5分钟。
    14. 吸出上清液。
    15. 将细胞核重悬于300μlPBS中。
      注意:此时,所有样品可以在4°C保存过夜,然后再继续步骤B.

  2. DNA提取
    1. 向细胞提取物中加入600μl冰冷的细胞裂解缓冲液。大部分提取物需要SDS降解;它不会影响最终的结果。彻底旋转30秒。
    2. 加入3μl蛋白酶K溶液(20 mg / ml)。在55℃孵育DNA样品3-16小时。时间取决于使用的样品和细胞系上注意到多少SDS沉淀。经验法则是更多的SDS沉淀和较大的细胞倾向于需要更多的孵育时间。
    3. 剪切DNA通过21 G针反复拉伸(15 x),避免过度起泡和气雾化损失。
    4. 将其冷却至室温,然后加入3μl4mg / ml无DNA酶的核糖核酸酶A.消化物在37℃下15-60分钟。
      注意:此时,所有样品可以在4℃下储存过夜,然后再进行到步骤B5。
    5. 酷到RT。加入200μl5 M醋酸钾;涡旋20秒
    6. 颗粒沉淀蛋白/ SDS通过在4℃下以最大RPM离心3分钟。
    7. 将溶液用600μl异丙醇转移到新鲜管中。通过倒置混合在最高转速下旋转3分钟。
    8. 去除上清液,用600μl70%乙醇洗涤DNA。以最大RPM旋转3分钟以沉淀颗粒。吸出乙醇。重复1分钟旋转并吸出剩余的EtOH。
    9. 空气干燥15分钟。
    10. 将沉淀重新溶解在100μl8mM NaOH或10mM Tris中。这可能很慢 - 建议在37℃孵育15分钟。
    11. DNA现在可以在-20°C储存,无事故。

  3. 评估NP-40分馏效率
    为了评估核 - 细胞质分离的准确性,可以使用qPCR,使用核,线粒体和全细胞提取物样品,如下列实施例。
    1. 将DNA样品在dH 2 O 2中稀释1至10,并在98℃下孵育20分钟,然后在冰上立即冷却。
    2. 每个PCR反应在15μl反应体积中含有7.5μlSYBR Green PCR Master混合物(Fermentas),2μl模板和1μM每种引物(表1)。

      表1. DNA扩增引物


    3. 使用ABI Prism 7900HT序列检测系统(PE-Applied Biosystems)对每个样品进行一式三份的QPCR进行扩增和检测。 PCR条件如下:

数据分析

  1. NP-40核 - 细胞质部分的准确性可以通过比较核部分中检测到的线粒体DNA拷贝的量和细胞质部分中β-珠蛋白DNA拷贝的量与在总细胞裂解物中发现的各个拷贝的量来验证。数据通过delta方法进行分析,需要平均阈值周期和标准偏差。因此,
    Ct =阈值循环,a =核或细胞质样品,b =全细胞裂解物样品:



  2. 将回收的产量与核级分中的细胞质污染相比较。图1改编自Galvis(2014)是可以用获得的数据组成的数据分析图的示例。线粒体DNA拷贝在核心部分与线粒体DNA拷贝在全细胞裂解物中的比例。该示例演示了成功的分馏。核部分核DNA与全细胞提取物的比例为1:1;而与整个细胞提取物相比,同时存在最小的线粒体DNA。与整个细胞提取物相比,细胞质级分含有最小的核DNA,而其中含有线粒体DNA与全细胞提取物的几乎1:1的比例。
  3. 请注意,推荐的包含数据是所有三倍体必须在彼此相隔一个周期内,所有获得的循环必须小于需要包含在qPCR运行中的阴性对照。


    图1.分馏方案的样品数据分析这是核和细胞质级分中的线粒体和β-珠蛋白DNA与总细胞裂解物的比例。用DBR1 shRNA pHyper-D4(D4)或DBR1 shRNA三重不匹配,pHyper-M4(M4)转染GHOST-R5X4细胞。二至二十四小时后,将细胞分成细胞核和细胞质级分,或裂解制备全细胞提取物。分离DNA用于qPCR以评估分级的成功。 A.与全细胞提取物相比,β-球蛋白引物用于测定细胞质级分中的核污染排除。该图示出了在整个细胞裂解物中细胞质级分中的β-珠蛋白拷贝与β-珠蛋白拷贝的比例。 B.与全细胞提取物相比,β-球蛋白引物用于测定核分数的浓度。该图示出了在全部细胞裂解物中核分数中β-珠蛋白拷贝与β-珠蛋白拷贝的比例。 C.线粒体引物用于测定细胞质分数与全细胞提取物相比的浓度。该图示出了在全部细胞裂解物中核分数中的线粒体DNA拷贝与β-珠蛋白拷贝的比例。 D.线粒体引物用于检测核提取物中线粒体污染的排除

笔记

  1. 方法C中提供的数据是Galvis等人于2014年提出的实验的未发表的对照。我们实验中的脱支酶1(DBR1)涉及HIV-1基因组的逆转录。
  2. 在步骤A中,步骤12,不要干扰沉淀物是非常重要的,因此最好留下一些上清液。后续步骤的目的是去除剩余的线粒体DNA。
  3. 在我们的经验中,与10mM Tris相比,用8mM NaOH重新悬浮DNA给下游应用提供更好的总体回收率。然而,如果所需的实验仅需要qPCR目的,而不需要测序,则10mM Tris足以使最终沉淀物再悬浮。

食谱

  1. 1M MgCl 2(500ml)
    将101.65g MgCl 2•6H 2 O加入到500ml ddH 2 O中, 在室温下存放
  2. 2.5 M蔗糖
    8.55克蔗糖 高达10毫升与ddH 2 O
    需要加热至37°C以使蔗糖完全溶解 在室温下存放
  3. TMK(10 ml;储存于4°C)


  4. TMK + 1%NP-40(2 ml)
    1.8 ml TMK
    200μl10%NP-40
    储存于4°C
  5. S1缓冲区
    0.25 M蔗糖 10mM MgCl 2


  6. S2缓冲区
    0.35 M蔗糖
    0.5mM MgCl 2
    储存于4°C


  7. 细胞裂解缓冲液
    10mM Tris-HCl,pH 7.4
    0.1%SDS
    1 mM EDTA
    储存于4°C

致谢

这项工作部分得到加州艾滋病毒/艾滋病研究计划的ID07-I-124授予David Camerini的支持。支持UCI病毒研究中心,UCI癌症研究所和ao族综合癌症中心。 Alvaro Galvis由UCI医学科学家培训计划支持,授予T32-GM08620。这个协议是从原来的Sambrook和Russell,2001修改和修改的。

参考

  1. Galvis,A.E。(2014)。 HIV-1 cDNA合成中的RNA系列中间体。 ProQuest 3615193。
  2. Galvis,A.E.,Fisher,H.E.,Nitta,T.,Fan,H.and Camerini,D。(2014)。 由DBR1敲低造成的HIV-1 cDNA合成的损害。 Vi Vi < 88(12):7054-7069。
  3. Sambrook,J.and Russell,D.W。(2001)。 分子克隆:实验手册。CSHL Press 。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Galvis, A., Fisher, H. E. and Camerini, D. (2017). NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells. Bio-protocol 7(20): e2584. DOI: 10.21769/BioProtoc.2584.
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Julia Guenther
Medical University of Innsbruck
Nice protocol! I will try it out in the coming weeks. I noticed, however, that in Procedure A - Steps 11 & 12 suggest the use of 500 ml. I am assuming you mean 500 µl. It that correct?
11/3/2017 5:51:56 AM Reply
Alvaro E. Galvis
Department of Molecular Biology & Biochemistry, University of California, USA, USA,

Yes, that's correct

3/4/2018 9:19:50 PM