搜索

Purification and Sequencing of DNA Guides from Prokaryotic Argonaute
原核Argonaute的DNA引导子的纯化和测序   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Some proteins utilize nucleic acids to guide them to complementary nucleic acid targets. One example is prokaryotic Argonaute protein, which, binds small single stranded DNA molecules as guides (Swarts et al., 2014). This protocol describes a method to purify DNA guides from these proteins. It also describes a PCR-based method to enrich the guides by PCR amplification. This methods relies on addition of a poly-A tail at the 3’-end of the ssDNA molecules by Terminal Deoxynucleotidyl Transferase (TdT), followed by ligation of a oligonucleotide to the 5’-end of the ssDNA molecule using T4 RNA ligase, and amplification by PCR. The generated dsDNA products are suitable for traditional cloning and sequencing and high-throughput sequencing. Importantly, the information which strand matches the ssDNA molecule is not lost during this process.

Materials and Reagents

  1. Purifying nucleic acids from proteins
    1. Purified protein and co-purified nucleic acids in purification buffer (e.g. TtAgo with siDNA guides)
      Note: Please refer to the protocol “Expression and Purification of the Thermus thermophilus Argonaute Protein” (Swarts et al., 2014b).
    2. Proteinase K solution (20 mg/ml) (Life Technologies, Ambion®, catalog number: AM2548 )
    3. CaCl2 solution (50 μM)
    4. Roti phenol/chloroform/isoamyl alcohol (pH 7.5-8.0) (Carl Roth, catalog number: A156 )
    5. 99% ethanol
    6. 70% ethanol (pre-cooled to -20 °C)
    7. Linear acrylamide (5 mg/ml) (Life Technologies, Ambion®, catalog number: AM9520 )
      Note: Alternatively, a house-made Linear Acrylamide can be used (http://www.uvm.edu/~tpdelane/lab/protocols/LinearPolyAcryl.htm).
    8. RNase free MQ water
    9. Additional materials and reagents required for analysis of purified nucleic acids (optional)
      1. T4 polynucleotide kinase (PNK) (New England Biolabs, catalog number M0201 )
      2. T4 polynucleotide kinase reaction buffer (New England Biolabs, catalog number M0201)

  2. Enriching and preparing ssDNA molecules for sequencing
    1. Purified single stranded DNA nucleotides in MQ water (e.g. see Procedure part A "purifying nucleic acids from proteins")
    2. RNase A (DNase and protease-free) (10 mg/ml) (Thermo Fisher Scientific, catalog number: EN0531 )
    3. Terminal Deoxynucleotidyl transferase (TdT) (recombinant) (Life Technologies, InvitrogenTM, catalog number: 10533-073 )
    4. Terminal Deoxynucleotidyl transferase (TdT) buffer (Life Technologies, InvitrogenTM, catalog number: 16314-015 )
    5. dATP (100 mM) (Thermo Fisher Scientific, catalog number: R0141 )
    6. QIAquick nucleotide removal kit (QIAGEN, catalog number: 28304 )
    7. Primer A (5’-GAGAGAGGATCCCGAATTGTGCAGCTGTCAATCAACC-3’) (5 μM)
    8. T4 RNA ligase 1 (New England BioLabs, catalog number: M0204 )
    9. T4 RNA ligase reaction buffer (10x, supplied with T4 RNA ligase 1)
    10. ATP (10 mM) (supplied with T4 RNA ligase 1)
    11. PEG8000 (supplied with T4 RNA ligase 1)
    12. Primer B (5’-GAGAGAGGATCCTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’) (5 μM)
    13. Pfu DNA polymerase (or equivalent)
    14. 10x Pfu DNA polymerase buffer (or equivalent, supplied with MgCl2 or MgSO4)
    15. dNTPs (5 μM)
    16. 2% agarose gel
    17. Generuler low range DNA ladder (Thermo Fisher Scientific)
    18. GeneJET gel extraction kit (Thermo Fisher Scientific, Fermentas, catalog number: K0691 )
      Note: Additional materials and reagents required only if ssDNA is not 5’-end phosphorylated: T4 polynucleotide kinase (PNK, New England Biolabs, catalog number: M0201) and T4 polynucleotide kinase reaction buffer (New England Biolabs, catalog number: M0201).

Equipment

  1. Purifying nucleic acids from proteins
    1. Heat block
    2. PCR machine
    3. Cooled table top Eppendorf centrifuge
    4. Freezer (-20 °C)

  2. Enriching and preparing ssDNA molecules for sequencing
    1. Heat block
    2. PCR machine
    3. Table top centrifuge
    4. Agarose gel electrophoresis equipment

Procedure

  1. Purifying nucleic acids from proteins
    1. Take purified protein sample (e.g. 50-500 µl, protein concentration up to 10 μM).
    2. Add CaCl2 to a final concentration of 5 µM.
    3. Add 1/10 volume of Proteinase K solution (e.g. to 450 µl protein/CaCl2 solution, add 50 µl Proteinase K solution).
    4. Incubate 4 h at 65 °C in a heat block. Alternatively, small volumes (up to 200 µl) can be incubated 4 h at 65 °C in a PCR machine.
    5. Add Roti phenol/chloroform/isoamyl alcohol (pH 7.5-8.0) in a 1:1 ratio (e.g. to 500 µl, add 500 µl phenol/chloroform/isoamyl alcohol).
    6. Vortex at max speed for 5 sec.
    7. Centrifuge the sample for 15 min in a table top centrifuge at maximum speed.
    8. Carefully pipet the upper fraction (aqueous phase containing the nucleic acids) to a clean centrifuge tube. Take less than the actual upper fraction (e.g. from a 500 μl sample, take only 450 μl). This avoids mixing with the lower fraction (organic phase containing proteins) which contains the phenol which might interfere with downstream steps. Discard the lower fraction.
    9. If the total volume of the upper fraction is larger than 500 µl, distribute the sample equally over multiple tubes.
    10. Add 99% ethanol in a 2:1 ratio (ethanol:sample).
    11. Add 10 µl linear polyacrylamide (5 µg/µl) as carrier.
    12. Incubate the samples at -20 °C for at least 16 h. Small nucleic acids take longer to precipitate.
    13. Centrifuge in a table top centrifuge at 4 °C for 30 min at maximum speed (13,000 rpm).
    14. Remove all ethanol with a pipette, a small white/transparent pellet should be visible-try not to disturb the pellet.
    15. Add 500 µl pre-cooled -20 °C, 70% ethanol to the pellet.
    16. Centrifuge in a table top centrifuge at 4 °C for 15 min at maximum speed.
    17. Remove all ethanol with a pipette-a small white/transparent pellet should be visible-try not to disturb the pellet.
    18. Incubate the reaction tube at 50 °C until all ethanol is evaporated (5-10 min).
    19. Resuspend the nucleic acids in 50 μl RNase-free MQ water.
    20. Analyze nucleic acids by 32P-labeling (see Figure 1) or use for enriching and preparing ssDNA molecules for sequencing (procedure B below). For 32P-labeling, use T4 polynucleotide kinase (T4 PNK) according to the protocol provided by the manufacturer.

  2. Enriching and preparing ssDNA molecules for sequencing
    1. Add 1 µl of DNase free RNase A to 50 µl purified ssDNA.
    2. Incubate at RT for 10 min.
    3. To polyadenylate the ssDNAs, use Terminal Deoxynucleotidyl transferase according to the protocol provided by the manufacturer.
      10 µl 5x TdT reaction buffer
      36.5 μl ssDNA (30-100 ng, if necessary, add MQ water)
      1.5 μl dATP (100 mM)
      1.5 μl TdT
    4. Mix and incubate for 60 min at 37 °C.
    5. Incubate for 10 min at 70 °C to inactivate TdT.
    6. Purify the samples using the QIAquick nucleotide removal kit according to the manual provided by the manufacturer. Elute in 30 μl MQ water.
      Notes:
      1. Other nucleic acid purification kits might also be suitable for purifying the nucleic acids. However, most nucleic acid purification kits do not purify nucleic acids <100 nt. Ensure the kit you use is suitable for the size of nucleic acid you are purifying.
      2. The next step is dependent on the presence of a phosphorylated 5’-end on your ssDNA molecules. If you are not sure if your small DNA molecule has a phosphorylated 5’-end, it is possible to phosphorylate it with T4 polynucleotide kinase according to the manual provided by the manufacturer. After 5’-end phosphorylation, purify the samples using the QIAquick nucleotide removal kit according to the manual provided by the manufacturer. Elute in 30 μl MQ water.
    7. Ligate primer A (see Notes a and e) to the phosphorylated 5’-ends of the ssDNA molecules with 3’-end poly A tails by mixing 16 μl ssDNA, 1 μl primer A, 6 μl ATP (10 mM), 6 μl 10x Reaction buffer, 30 μl 50% PEG8000 (w/v) and 1 μl T4 RNA ligase. Incubate for 16 h at 25 °C. T4 RNA ligase is used as it can ligate a 3’-end of a ssDNA strand (the primer) to a 5’-end phosphorylated ssDNA strand (the ssDNA molecules with 3’-end poly A tails).
    8. Purify the samples using the QIAquick nucleotide removal kit according to the manual provided by the manufacturer. Elute in 30 μl MQ water.
    9. To enrich the ssDNA molecules, PCR amplify the ssDNA molecule using primers A and B.
      2 μl 10x Pfu polymerase buffer (containing MgCl2 or MgSO4)
      14 μl ssDNA with 5’ oligonucleotide and 3’ poly A tail
      1 μl primer A (5 μM)
      1 μl primer B (5 μM)
      1 μl dNTPs (5 μM)
      1 μl Pfu polymerase
    10. Run a PCR
      1 min at 95 °C
      ----------
      20 cycles:
      1 min at 95 °C (denaturation)
      30 sec at 40 °C (annealing)
      1 min at 72 °C (elongation)
      ----------
      1 min at 72 °C
      Notes:
      1. The annealing temperature is dependent on your primers. The elongation time is dependent on the DNA that you want to amplify. The amount of cycles required is dependent on the amount of template and the amount of product required. Generally, less cycles is better for dsDNA quality, while more cycles generate more dsDNA fragments.
      2. Run the PCR product on a 2% agarose gel.
      3. Sometimes two (or more) bands appear after the PCR. The lowest (smallest) band often corresponds with a PCR product containing only the primers but no DNA of interest. Based on the size of your co-purified ssDNA, you should be able to predict the expected size of a successful generated and amplified dsDNA product (see Figure 2). Cut out the band of the correct size and purify the dsDNA fragments from this band using the GeneJET gel extraction kit according to the protocol provided by the manufacturer.
      4. If the yield of the dsDNA molecules is too low for cloning/sequencing, the PCR can be repeated on the dsDNA purified from the gel. Ensure to repeat the gel purification, as also products of other lengths might be amplified.
      5. The purified dsDNA PCR products can be sequenced directly by a modified Illumina sequencing protocol. In order to do this, ensure that primer A contains a sequence suitable for the sequencing platform used (discuss this with your sequencing company). Alternatively, the purified dsDNA PCR products can be cloned in a vector using the introduced KpnI restriction sites on either end of the PCR product (or other restriction sites, if you designed your own primers). Alternatively, the products can be cloned by TOPO cloning (Life Technologies) after addition of 3’-end adenine groups with Taq polymerase (see protocol provided by the manufacturer). After transformation of these vectors, plasmids can be purified and sequenced using standard protocols. After sequencing, the strand originating from the single stranded molecule can be identified by the presence of the 5’-end Primer A sequence and 3’-end poly-A sequence.

Representative data


Figure 1. 20% denaturing polyacrylamide gels with resolved nucleic acids that co-purified with TtAgo or TtAgoDM (a catalytic mutant of TtAgo, unable to acquire guides in vivo) (Swarts et al., 2014a). Nucleic acids were 32P labelled by replacing 5’-phosphate (left gel) or 5’-OH groups (right gel) with 32P using T4 polynucleotide kinase. Labelled nucleic acids were cleaved with RNase A, DNase I or nuclease P1 (cleaves ssRNA and ssDNA). The left gel shows that TtAgo co-purified with small ssDNA molecules with a 5’-end phosphate. The right gel shows that both TtAgo and TtAgoDM co-purify with long ssRNA molecules of undefined length. Figure adapted from Swarts et al. (2014a).


Figure 2. 2% agarose gel with resolved enriched dsDNA molecules. M: Generuler low range DNA ladder. Sizes corresponding with the DNA in the marker lane are indicated in bp. Lanes 1 and 2 contain generated dsDNA samples to which 21 nt ssDNA was added in step B1 of the protocol. Lanes 3 and 4 contain negative controls with generated dsDNA molecules consisting of only the oligonucleotide and 3’-end poly A tail without an ssDNA insert. Note that sometimes, bands representing both molecules with and without an insert will appear in the sample of interest. In this case, make sure you select the right band (usually the upper band, as it contains an insert and thus is larger) for further processing and sequencing.

Acknowledgments

This work was financially supported by a grant from the Netherlands Organization of Scientific Research (NWO) to J.O. (NWO-TOP, 854.10.003).

References

  1. Swarts, D. C., Jore, M. M., Westra, E. R., Zhu, Y., Janssen, J. H., Snijders, A. P., Wang, Y., Patel, D. J., Berenguer, J., Brouns, S. J. and van der Oost, J. (2014a). DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507(7491): 258-261.
  2. Swarts, D. C., Jore, M. M. and van der Oost, J. (2014b). Expression and purification of the Thermus thermophilus argonaute protein. Bio-protocol 4(19): e1253.

简介

一些蛋白利用核酸来将它们引导至互补核酸靶。 一个实例是原核Argonaute蛋白,其结合小的单链DNA分子作为指导(Swarts等人,2014)。 该协议描述了从这些蛋白质中纯化DNA指南的方法。 它还描述了基于PCR的方法以通过PCR扩增来富集指南。 该方法依赖于通过末端脱氧核苷酸转移酶(TdT)在ssDNA分子的3'末端添加聚腺苷酸尾,然后使用T4 RNA连接酶将寡核苷酸连接到ssDNA分子的5'末端, 和通过PCR扩增。 所产生的dsDNA产物适合于传统的克隆和测序以及高通量测序。 重要的是,与ssDNA分子匹配的链的信息在该过程中不会丢失。

材料和试剂

  1. 从蛋白质纯化核酸
    1. 在纯化缓冲液中纯化的蛋白质和共纯化的核酸(例如 Tt Ago与siDNA指南)
      注意:请参阅协议"表达和纯化 嗜热栖热菌Argonaute蛋白"(Swarts等人,2014b)。
    2. 蛋白酶K溶液(20mg/ml)(Life Technologies,Ambion ,目录号:AM2548)
    3. CaCl 2溶液(50μM)
    4. Roti苯酚/氯仿/异戊醇(pH 7.5-8.0)(Carl Roth,目录号:A156)
    5. 99%乙醇
    6. 70%乙醇(预冷至-20℃)
    7. 线性丙烯酰胺(5mg/ml)(Life Technologies,Ambion ,目录号:AM9520)
      注意:或者,可以使用自制的线性丙烯酰胺 ( http://www.uvm.edu/~tpdelane/lab/protocols/LinearPolyAcryl.htm )。
    8. RNase游离MQ水
    9. 分析纯化核酸所需的其他材料和试剂(可选)
      1. T4多核苷酸激酶(PNK)(New England Biolabs,目录号M0201)
      2. T4多核苷酸激酶反应缓冲液(New England Biolabs,目录号M0201)

  2. 富集和准备ssDNA分子进行测序
    1. 在MQ水中纯化的单链DNA核苷酸(例如参见程序部分A"从蛋白质纯化核酸")
    2. RNA酶A(DNase和无蛋白酶)(10mg/ml)(Thermo Fisher Scientific,目录号:EN0531)
    3. 末端脱氧核苷酸转移酶(TdT)(重组体)(Life Technologies,Invitrogen TM ,目录号:10533-073)
    4. 末端脱氧核苷酸转移酶(TdT)缓冲液(Life Technologies,Invitrogen TM ,目录号:16314-015)
    5. dATP(100mM)(Thermo Fisher Scientific,目录号:R0141)
    6. QIAquick核苷酸去除试剂盒(QIAGEN,目录号:28304)
    7. 引物A(5'-GAGAGAGGATCCCGAATTGTGCAGCTGTCAATCAACC-3')(5μM)
    8. T4 RNA连接酶1(New England BioLabs,目录号:M0204)
    9. T4 RNA连接酶反应缓冲液(10×,用T4 RNA连接酶1提供)
    10. ATP(10mM)(用T4 RNA连接酶1提供)
    11. PEG8000(T4 RNA连接酶1提供)
    12. 引物B(5'-GAGAGAGGATCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3')(5μM)
    13. Pfu DNA聚合酶(或同等物)
    14. 10x Pfu DNA聚合酶缓冲液(或等同物,提供MgCl 2或MgSO 4)
    15. dNTPs(5μM)
    16. 2%琼脂糖凝胶
    17. Generator低范围DNA梯形图(Thermo Fisher Scientific)
    18. GeneJET凝胶提取试剂盒(Thermo Fisher Scientific,Fermentas,目录号:K0691)
      注意:只有ssDNA不需要时,才需要其他材料和试剂 5'-末端磷酸化:T4多核苷酸激酶(PNK,New England Biolabs,目录号:M0201)和T4多核苷酸激酶反应 缓冲液(New England Biolabs,目录号:M0201)

设备

  1. 从蛋白质纯化核酸
    1. 热块
    2. PCR机
    3. 冷却台面Eppendorf离心机
    4. 冷冻(-20°C)

  2. 富集和准备ssDNA分子进行测序
    1. 热块
    2. PCR机
    3. 台式离心机
    4. 琼脂糖凝胶电泳设备

程序

  1. 从蛋白质纯化核酸
    1. 取纯化的蛋白质样品(例如50-500μl,蛋白质浓度高达10μM)。
    2. 加入CaCl 2至终浓度为5μM。
    3. 加入1/10体积的蛋白酶K溶液(例如至450μl蛋白/CaCl 2溶液,加入50μl蛋白酶K溶液)。
    4. 在65℃在加热块中孵育4小时。 或者,小体积 (高达200μl)可以在65℃下在PCR仪中孵育4小时。
    5. 以1:1的比例加入Roti苯酚/氯仿/异戊醇(pH 7.5-8.0) (例如加至500μl,加入500μl苯酚/氯仿/异戊醇)。
    6. 以最大速度涡旋5秒。
    7. 在台式离心机中以最大速度离心样品15分钟
    8. 小心吸取上部分(含水相的水相) 核酸)至干净的离心管。 小于实际 (例如来自500μl样品的部分,仅需450μl)。 这个 避免与低级部分(含有机相的混合物)混合 蛋白质),其含有可能干扰的酚 下游步骤。 舍弃较低分数。
    9. 如果上部分的总体积大于500μl,则将样品平均分布在多个管上。
    10. 以2:1的比例(乙醇:样品)加入99%乙醇。
    11. 加入10微升线性聚丙烯酰胺(5微克/微升)作为载体
    12. 将样品在-20°C孵育至少16小时。 小核酸需要更长时间才能沉淀
    13. 在台式离心机中在4℃以最大速度(13,000rpm)离心30分钟
    14. 用移液管除去所有乙醇,应该看到一个小白色/透明的小丸 - 尽量不要打扰小丸
    15. 加入500μl预冷-20°C,70%乙醇的沉淀。
    16. 在台式离心机中以4℃以最大速度离心15分钟
    17. 用移液管除去所有乙醇 - 应看到一个小白色/透明的小丸 - 尽量不要打扰小丸
    18. 在50℃下孵育反应管,直到所有乙醇蒸发(5-10分钟)
    19. 重悬在50微升无RNA酶MQ水中的核酸。
    20. 通过 P标记(参见图1)或使用for分析核酸 富集和制备用于测序的ssDNA分子(方法B 下面)。 对于 32 P标记,使用T4多核苷酸激酶(T4 PNK) 根据制造商提供的协议。

  2. 富集和准备ssDNA分子进行测序
    1. 加入1微升无DNA酶的RNase A到50微升纯化的ssDNA
    2. 在RT孵育10分钟。
    3. 为了多腺苷酸化ssDNA,使用末端脱氧核苷酸 转移酶根据制造商提供的协议 10μl5x TdT反应缓冲液
      36.5μlssDNA(30-100ng,如有必要,加入MQ水)
      1.5μldATP(100mM) 1.5μlTdT
    4. 混合并在37℃下孵育60分钟
    5. 在70°C孵育10分钟以灭活TdT。
    6. 使用QIAquick核苷酸去除试剂盒纯化样品 根据制造商提供的手册。 在30μlMQ中洗脱 水。
      注意:
      1. 其他核酸纯化试剂盒可能 也适合于纯化核酸。 然而,大多数核酸 酸纯化试剂盒不纯化小于100nt的核酸。 确保 您使用的套件适合您的核酸大小 净化。
      2. 下一步取决于a的存在 磷酸化的5'末端。 如果你不确定是否 你的小DNA分子有一个磷酸化的5'端,这是可能的 根据手册用T4多核苷酸激酶磷酸化它 由制造商提供。 5'末端磷酸化后,纯化 样品使用QIAquick核苷酸去除试剂盒 手册由制造商提供。 在30μlMQ水中洗脱。
    7. 将磷酸化的5'-末端引物引物A(参见注释a和e)   ssDNA分子与3'端poly A尾通过混合16微升ssDNA,1微升 引物A,6μlATP(10mM),6μl10x反应缓冲液,30μl50%PEG8000 (w/v)和1μlT4 RNA连接酶。 在25°C孵育16小时。 T4 RNA连接酶 因为其可以将ssDNA链(引物)的3'-末端连接到a 5'-末端磷酸化的ssDNA链(ssDNA分子具有3'-末端聚   A tails)。
    8. 使用QIAquick核苷酸纯化样品 拆卸套件根据制造商提供的手册。 洗脱 在30μlMQ水中
    9. 为了富集ssDNA分子,使用引物A和B扩增ssDNA分子 2μl10x Pfu聚合酶缓冲液(含有MgCl 2或MgSO 4)
      14μl具有5'寡核苷酸和3'多聚A尾的ssDNA
      1μl引物A(5μM)
      1μl引物B(5μM)
      1μldNTPs(5μM)
      1μlPfu聚合酶
    10. 运行PCR
      95℃1分钟
      ----------
      20个周期:
      95℃1分钟(变性)
      40℃下30秒(退火)
      72℃1分钟(伸长)
      ----------
      72℃1分钟
      注意:
      1. 退火温度取决于你的引物。 的 延伸时间取决于你想扩增的DNA。 的 所需的循环量取决于模板的量和   所需产品数量。 通常,较少的循环对于dsDNA是更好的 质量,而更多的循环产生更多的dsDNA片段。
      2. 在2%琼脂糖凝胶上运行PCR产物。
      3. 有时在PCR之后出现两个(或更多)条带。 最低的 (最小)带通常对应于仅含有的PCR产物   但不含目标DNA。 基于您的共纯化的大小 ssDNA,你应该能够预测成功的预期大小 产生和扩增的dsDNA产物(参见图2)。 切出乐队 并从该条带中纯化dsDNA片段 GeneJET凝胶提取试剂盒按照提供的方案   制造商。
      4. 如果dsDNA分子的产量太低,   克隆/测序,可以对从纯化的dsDNA重复PCR 凝胶。 确保重复凝胶纯化,也作为产品 其他长度可能会放大。
      5. 纯化的dsDNA PCR产物   可以通过修改的Illumina测序方案直接测序。 为此,确保引物A含有合适的序列 对于所使用的测序平台(与您的测序讨论这一点 公司)。 或者,可以克隆纯化的dsDNA PCR产物 在使用在任一端引入的KpnI限制性位点的载体中   PCR产物(或其他限制性位点,如果您设计了自己的 引物)。 或者,可以通过TOPO克隆来克隆产物 (Life Technologies)在用Taq加入3'-末端腺嘌呤基团后 聚合酶(参见制造商提供的方案)。 后 转化这些载体,可以纯化质粒并测序 使用标准协议。 测序后,来自的链 单链分子可以通过其存在来鉴定 5'-末端引物A序列和3'-末端聚腺苷酸序列。

代表数据


图1.具有 Tt Ago或 共同纯化的解析的核酸的20%变性聚丙烯酰胺凝胶> Tt ,2014a)。核酸是通过用 32 取代5'-磷酸(左凝胶)或5'-OH基团(右凝胶)标记的< P使用T4多核苷酸激酶。标记的核酸用核糖核酸酶A,DNA酶I或核酸酶P1切割(切割ssRNA和ssDNA)。左凝胶显示用小的ssDNA分子与5'-末端磷酸共纯化的Tt Ago。正确的凝胶显示,Tt Ago和 AgoDM与未定义长度的长ssRNA分子共同纯化。图改编自Swarts等人。 (2014a)。


图2.具有溶解的富集dsDNA分子的2%琼脂糖凝胶。 M:Generator低范围DNA梯。对应于标记物泳道中的DNA的大小以bp表示。泳道1和2含有产生的dsDNA样品,在该方案的步骤B1中向其中加入21nt ssDNA。泳道3和4包含具有仅由寡核苷酸组成的生成的dsDNA分子和没有ssDNA插入片段的3'-端聚腺苷酸尾的阴性对照。注意,有时,表示具有和不具有插入物的分子的条带将出现在感兴趣的样品中。在这种情况下,请确保选择正确的谱带(通常是上谱带,因为它包含插入片段,因此较大),以进一步处理和测序。

致谢

这项工作得到了荷兰科学研究组织(NWO)给J.O.的资助。 (NWO-TOP,854.10.003)。

参考文献

  1. Swarts,DC,Jore,MM,Westra,ER,Zhu,Y.,Janssen,JH,Snijders,AP,Wang,Y.,Patel,DJ,Berenguer,J.,Brouns,SJand van der Oost, 2014a)。 由原核生物Argonaute进行的DNA引导的DNA干扰。 自然 507(7491):258-261。
  2. Swarts,D.C.,Jore,M.M.and van der Oost,J.(2014b)。 嗜热栖热菌(Thermus thermophilus)的表达和纯化 argonaute蛋白。 生物协议 4(19):e1253。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Swarts, D. C., Westra, E. R., Brouns, S. J. and van der Oost, J. (2014). Purification and Sequencing of DNA Guides from Prokaryotic Argonaute. Bio-protocol 4(22): e1293. DOI: 10.21769/BioProtoc.1293.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。