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[Bio101] Standard DNA Cloning
[Bio101] 标准DNA克隆   

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Abstract

This protocol describes general cloning steps from preparation of both vector and insert DNA to the ligation reaction.

Materials and Reagents

  1. Luria-Bertani broth (LB) medium: Bacto-tryptone (BD Biosciences), yeast extract (BD Biosciences)
  2. Antibiotics (Sigma-Aldrich/Thermo Fisher Scientific)
  3. QIAGEN Plasmid Purification Handbook (QIAGEN)
  4. SeaKem® LE Agarose (Cambrex)
  5. Plasmid Prep Kit (QIAGEN /Fermentas)
  6. PCR Clean-up kit (QIAGEN /Fermentas)
  7. Restriction enzymes (New England Biolabs)
  8. Alkaline Phosphatase: Calf intestinal alkaline phosphatase (CIP) (New England Biolabs, catalog number: M0290) or Shrimp Alkaline Phosphatase (SAP) (Promega Corporation, catalog number: M8201 )
  9. Ligase enzyme (New England Biolabs)
  10. DNA ladder
  11. NaCl
  12. LB broth media (see Recipes)
  13. Ligation reaction (see Recipes)

Equipment

  1. Nanodrop (Thermo Scientific)

Procedure

  1. Preparing vector DNA for cloning:
    Depending on the copy number of the vector plasmid, decide if you need the Mini-prep, Midi-prep, or Maxi-prep kit. If it is a high copy (>10 copies/cell) plasmid, plasmid DNA can be prepared by using the Mini-prep kit. If it is a low copy (<10 copies/cell) plasmid, use the Midi-prep or Maxi-prep kit.
    1. Grow E. coli cell culture carrying vector plasmid in LB liquid medium with appropriate antibiotics at 37 °C overnight.
    2. Follow QIAGEN Plasmid Purification Handbook to obtain DNA. If plasmid DNA does not need to be purified, and to be more economical, plasmid DNA can be extracted without using a plasmid prep kit (See protocol “Plasmid DNA extraction from E. coli using alkaline lysis method”).
    3. Estimate plasmid DNA concentration using one of the following two ways:
      1. Load 2-3 μl plamid DNA and a DNA ladder on a DNA agarose gel and estimate DNA according to the DNA marker.
      2. Easier and more accurate way is to measure DNA using Nanodrop if it is available.
    4. Digest 2-5 μg vector DNA using restriction enzymes needed for the insert DNA. To make sure the vector is completely digested, extra enzyme and long incubation may be needed.
    5. To reduce the chance of self-ligation, dephosphorylate the 5′ phosphorylated ends of the digested vector with alkaline phosphatase.
      Note: If the  shrimp alkaline phosphatase (SAP) is used, then add 2 μl SAP directly to 100 μl digest solution, incubate at 37 °C for 1 h, then inactivate SAP at 65 °C for 10 min. If the calf intestinal alkaline phosphatase (CIP) is used, then add 5 μl CIP enzyme to 100 μl digestion solution, incubate at 37 °C for 1 h, then inactivate SAP at 65 °C for 30 min.
    6. Perform gel purification of digested vector DNA.

  2. Preparing insert DNA for cloning:
    1. Obtain insert DNA from digestion of plasmid DNA.
      1. Extract plasmid DNA as described above.
      2. Digest plasmid DNA with appropriate restriction enzymes.
      3. Perform gel purification of insert DNA.
    2. Generate insert DNA from PCR product.
      1. Design primers using a free a good quality program online (e.g. http://frodo.wi.mit.edu/primer3/) containing desired cloning sites with several of bases flanking their recognition sequences (http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/ cleavage_olignucleotides.asp).
      2. Amplify insert DNA from a template by PCR, and clean up PCR product by PCR clean-up kit.
      3. Digest PCR product with the corresponding restriction enzymes. Or, first clone PCR product to pGEM T-easy vector, and then generate insert DNA from the resulting plasmid.
      4. Perform gel purification of insert DNA.
      5. Estimate DNA concentration.

  3. Ligation of insert and vector:
    1. Usually (particularly for blunt end ligation), need more insert DNA than vector: 1 mole of vector normally needs 5 or more moles of insert (see protocol “DNA molecular weight calculation”).
    2. Control ligation: To determine background clones arising from self-ligation of inefficiently phosphatased vector, set a parallel ligation in the absence of insert DNA.

  4. Transform 1 μl ligation reaction to competent cell by electroporation or chemical method.

  5. Colony PCR to screen for plasmids carrying the correct inserts and then confirm the result by digestion and sequencing of the plasmid.

Recipes

  1. 1 liter of LB broth media
    10 g Bacto-tryptone
    5 g yeast extract
    10 g NaCl
    Add ddH2O to get volume 1 L
    Sterilize by autoclaving.
  2. Ligation reaction
    X μl DNA vector( ~20 ng)
    Y μl insert (~100-1,000 ng)
    2 μl 10x buffer
    1 μl T4 DNA ligase
    To 20 μl H2O
    --------
    20 μl total

简介

该方案描述从载体和插入DNA制备到连接反应的一般克隆步骤。

材料和试剂

  1. Luria-Bertani肉汤(LB)培养基:细菌胰蛋白胨(BD Biosciences),酵母提取物(BD Biosciences)
  2. 抗生素(Sigma-Aldrich/Thermo Fisher Scientific)
  3. QIAGEN质粒纯化手册(QIAGEN)
  4. SeaKem ® LE琼脂糖(Cambrex)
  5. 质粒制备试剂盒(QIAGEN/Fermentas)
  6. PCR纯化试剂盒(QIAGEN/Fermentas)
  7. 限制酶(New England Biolabs)
  8. 碱性磷酸酶:小牛肠碱性磷酸酶(CIP)(New England Biolabs,目录号:M0290)或虾碱性磷酸酶(SAP)(Promega Corporation,目录号:M8201)
  9. 连接酶(New England Biolabs)
  10. DNA梯子
  11. NaCl
  12. LB肉汤培养基(见配方)
  13. 连接反应(参见配方)

设备

  1. Nanodrop(Thermo Scientific)

程序

  1. 准备载体DNA克隆:
    根据载体质粒的拷贝数,决定是否需要Mini-prep,Midi-prep或Maxi-prep试剂盒。如果它是高拷贝(> 10拷贝/细胞)质粒,则可以使用Mini-prep试剂盒制备质粒DNA。如果它是低拷贝(<10拷贝/细胞)质粒,使用Midi-prep或Maxi-prep试剂盒。
    1. 成长。大肠杆菌细胞培养物携带载体质粒在含有适当抗生素的LB液体培养基中在37℃过夜。
    2. 按照QIAGEN Plasmid Purification Handbook,获得DNA。如果质粒DNA不需要纯化,并且更经济,可以不使用质粒制备试剂盒提取质粒DNA(参见方案"使用碱性裂解法从大肠杆菌中提取质粒DNA ")。
    3. 使用以下两种方法之一估计质粒DNA浓度:
      1. 在DNA琼脂糖凝胶上加载2-3μlplamid DNA和DNA ladder,根据DNA标记估计DNA
      2. 更容易和更准确的方法是使用Nanodrop测量DNA,如果可用
    4. 使用插入DNA所需的限制酶消化2-5μg载体DNA。 为了确保载体完全消化,可能需要额外的酶和长时间的温育
    5. 为了降低自连接的可能性,用碱性磷酸酶使消化的载体的5'磷酸化末端脱磷酸。 注意:如果  使用虾碱性磷酸酶(SAP),然后将2μlSAP直接加入到100μl消化溶液中,在37℃孵育1小时,然后在65℃灭活SAP 10分钟。 如果使用小牛肠碱性磷酸酶(CIP),然后加入5μlCIP酶到100μl消化溶液中,在37℃孵育1小时,然后在65℃灭活SAP 30分钟。
    6. 进行消化的载体DNA的凝胶纯化

  2. 准备用于克隆的插入DNA:
    1. 从质粒DNA的消化获得插入DNA。
      1. 如上所述提取质粒DNA
      2. 用适当的限制酶消化质粒DNA
      3. 进行插入DNA的凝胶纯化
    2. 从PCR产物产生插入DNA。
      1. 使用免费的优质在线课程设计引文(例如 http://frodo.wi.mit.edu/引物3/),其含有具有其识别序列侧翼的几个碱基的所需克隆位点( http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp )。
      2. 通过PCR从模板扩增插入DNA,并通过PCR清除试剂盒清除PCR产物
      3. 用相应的限制酶消化PCR产物。 或者,首先克隆PCR产物到pGEM T-easy载体,然后从所得质粒产生插入DNA
      4. 进行插入DNA的凝胶纯化
      5. 估计DNA浓度。

  3. 插入物和载体的连接:
    1. 通常(特别是对于平端连接),比载体需要更多的插入DNA:1摩尔载体通常需要5或更多个插入片段(参见方案" DNA分子量计算")。
    2. 对照连接:为了确定由低效磷酸酶载体的自身连接产生的背景克隆,在不存在插入DNA的情况下设定平行连接。

  4. 通过电穿孔或化学方法转化1μl连接反应到感受态细胞
  5. 菌落PCR筛选携带正确插入片段的质粒,然后通过质粒的消化和测序确认结果。

食谱

  1. 1升LB肉汤培养基
    10克细菌用胰蛋白胨 5g酵母提取物
    10克NaCl
    添加ddH 2 O即可获得卷1 L
    通过高压灭菌消毒。
  2. 结扎反应
    XμlDNA载体(〜20ng)
    Yμl插入(〜100-1,000 ng)
    2μl10x缓冲液
    1μlT4 DNA连接酶
    加入20μlH 2 O 2 / --------
    总共20μl
  • English
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:He, F. (2011). Standard DNA Cloning. Bio-protocol Bio101: e52. DOI: 10.21769/BioProtoc.52;
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Bioseeker B
Unknown
Hello. I didn't understand step 6 (gel purification). My other question is that how we should know how many microliters we should add in any step of any molecular based experiment? Should we always have a protocol? What if the amounts differ by protocols?

Thanks.
7/29/2016 6:32:59 AM Reply
Fanglian He
Department of Biology, University of Pennsylvania, USA

Hi, Regarding gel purification, any commercial kit of gel purification (i.e.
QIAquick Gel Extraction Kit ) can be used.
For your second question, not sure what you meant by "any molecular based experiment". But, the amount of each reagent presented in this protocol worked in my hand. Of course, you could adjust the amount of each reagent accordingly if the volume of your reaction assay is different.

8/1/2016 2:18:31 PM


Alexander Westbye
Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, Netherlands
Less expensive plasmid miniprep (column based).
Most plasmid miniprep silica-column kits are expensive, however the costs of column-based minipreps can be significantly reduced by purchasing columns separately and making your own solutions.
One such company is Epoch Life Sciences which sells EconoSpin columns (#1920-050/250). The lab I work in has used this column with success for several years doing minipreps, PCR cleanups and the occasional DNA agarose-gel extraction. The product was supplied with recipes for the solutions.
3/19/2014 12:21:08 PM Reply
Marcin Wegrecki
IBV-CSIC

Alexander, could you please provide the recipes for the the solutions. In my lab we've been using them for the last year but we can't find the sheet that came with the columns to prepare more buffers. They don't share it on the manufacturer website anymore... Thanks a lot.

10/2/2014 10:06:47 AM


Cherysa Anselm
University of the West Indies
What is the chemical method to transform staphylococcus aureus?
6/19/2013 4:27:02 PM Reply
Yuanqing Lin
Assalamoalaikum
i have a question regardind ligation reaction
atually m facing problem with selfligation so i need a protocol for ligation of insert in vector by using alkaline phosphatase. how much vol. of this enzyme should be used in ligation reaction of final volume 30ul.
5/14/2011 2:59:21 PM Reply
Fanglian He
Department of Biology, University of Pennsylvania, USA

Treat vector with alkaline phosphatase before set up the ligation reaction. Please see this link http://www.bio-protocol.com/wenzhang.aspx?id=43&fl3=7 for protocol of using alkaline phosphatase.

8/31/2011 3:44:06 PM