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Standard DNA Cloning
Published: Apr 5, 2011 DOI: 10.21769/BioProtoc.52 Views: 65567
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Abstract
This protocol describes general cloning steps from preparation of both vector and insert DNA to the ligation reaction.
Background
Materials and Reagents
- Luria-Bertani broth (LB) medium: Bacto-tryptone (BD Biosciences), yeast extract (BD Biosciences)
- Antibiotics (Sigma-Aldrich/Thermo Fisher Scientific)
- QIAGEN Plasmid Purification Handbook (QIAGEN)
- SeaKem® LE Agarose (Cambrex)
- Plasmid Prep Kit (QIAGEN /Fermentas)
- PCR Clean-up kit (QIAGEN /Fermentas)
- Restriction enzymes (New England Biolabs)
- Alkaline Phosphatase: Calf intestinal alkaline phosphatase (CIP) (New England Biolabs, catalog number: M0290) or Shrimp Alkaline Phosphatase (SAP) (Promega Corporation, catalog number: M8201 )
- Ligase enzyme (New England Biolabs)
- DNA ladder
- NaCl
- LB broth media (see Recipes)
- Ligation reaction (see Recipes)
Equipment
- Nanodrop (Thermo Scientific)
Procedure
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
Category
Molecular Biology > DNA > DNA cloning
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