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Standard DNA Cloning

Molecular Biology > DNA > DNA cloning
Author: Fanglian He
Vol 1, Iss 7, 4/5/2011, 11153 views, 3 Q&A

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

Materials and Reagents

  1. Luria-Bertani broth (LB) medium: Bacto-tryptone (BD), yeast extract (BD)
  2. Antibiotics (Sigma/Fisher)
  3. SeaKem® LE Agarose (Cambrex)
  4. Plasmid Prep Kit (Qiagen/Fermentas)
  5. PCR Clean-up kit (Qiagen/Fermentas)
  6. Restriction enzymes (NEB)
  7. Alkaline Phosphatase: Calf intestinal alkaline phosphatase (CIP) (NEB, catalog number: M0290) or Shrimp Alkaline Phosphatase (SAP) (Promega, catalog number: M8201) Ligase enzyme (NEB)

Equipment

  1. Nanodrop (Thermo Scientific)

Procedure

  1. Preparing Vector DNA for Cloning :
    Depend on copy number of vector plasmid to decide if you need Mini-prep, Midi-prep, or Maxi-prep kit. If it is high copy (> 10 copies/cell) plasmid, plasmid DNA can be prepared by using Mini-prep kit. If it is low copy (< 10 copies/cell) plasmid, use Midi-prep, or Maxi-prep.
    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 save money, plasmid DNA can be extracted without using plasmid prep kit (See Protocol “Plasmid DNA extraction from E. coli using alkaline lysis method”)
    3. Estimate plasmid DNA concentration: 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 insert DNA. To make sure the vector is completely digested, extra enzyme and long incubation may be needed.
    5. To reduce chance of self-ligation, dephosphorylate the 5′ phosphorylated ends of digested vector with Alkaline Phosphatase.
      Note: If use Shrimp Alkaline Phosphatase (SAP). Usually, 2 μl SAP is added directly to 100 μl digest solution, incubate at 37 °C for 1 h, and then inactivate SAP at 65 °C for 10 min; If use Calf intestinal alkaline phosphatase (CIP),add 5 μl CIP enzyme to 100 μl digestion solution, incubate at 37 °C for 1 h, and then inactivate CIP at 65 °C for 30 min.
    6. 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. Gel purification of insert DNA
    2. Generate insert DNA from PCR product
      1. Design primers using a free program online[1] containing desired cloning sites with a several of bases flanking their recognition sequences[2]
      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. 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 the 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 dH2O 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

References

  1. http://frodo.wi.mit.edu/primer3/
  2. http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/ cleavage_olignucleotides.asp


How to cite this protocol: He, F. (2011). Standard DNA Cloning. Bio-protocol 1(7): e52. http://www.bio-protocol.org/e52



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