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[Bio101] Standard DNA Cloning

Molecular Biology > DNA > DNA cloning
Author: Fanglian He
4/5/2011, 16137 views, 3 Q&A

[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


How to cite this protocol: (2011). Standard DNA Cloning. Bio-protocol Bio101: e52. http://www.bio-protocol.org/e52



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Anonymous

5/14/2011 2:59:21 PM  

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.

Fanglian

8/31/2011 3:44:06 PM  

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.

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Cherysa

6/19/2013 4:27:02 PM  

What is the chemical method to transform staphylococcus aureus?

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Alexander

3/19/2014 12:21:08 PM  

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.

Marcin

10/2/2014 10:06:47 AM  

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.

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