Ligation of Linkers or Adapters to Double‐Stranded cDNA

Lloyd B. Klickstein1, Rachael L. Neve2

1 Brigham and Women's Hospital, Belmont, Massachusetts, 2 McLean Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 5.6
DOI:  10.1002/0471142727.mb0506s13
Online Posting Date:  May, 2001
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Abstract

The describes the strategy of producing a genomic DNA library. A number of small‐scale ligations are performed using a set amount of vector and varying amounts of insert. Test ligations are transformed into bacteria (plasmid vectors) or packaged and plated on host bacteria (λ and cosmid vectors). The number of clones in the different ligations is compared, and the optimum ratio of vector to insert is indicated by the ligation with the most recombinant clones. A large‐scale ligation is then set up using this optimum ratio. This protocol employs a bacteriophage vector; however, cosmid or plasmid vectors can be used with minor modifications.

     
 
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Table of Contents

  • Basic Protocol 1: Methylation of cDNA and Ligation of Linkers
  • Alternate Protocol 1: Ligation of BstXI Synthetic Adapters
  • Support Protocol 1: Preparation of a CL‐4B Column
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Methylation of cDNA and Ligation of Linkers

  Materials
  • Blunt‐ended, double‐stranded radiolabeled cDNA (unit 5.5)
  • 2× methylase buffer (Table 97.80.4711)
  • recipe50× S‐adenosylmethionine (SAM), freshly prepared (or from New England Biolabs with order of methylase)
  • EcoRI methylase ( New England Biolabs; unit 3.1)
  • TE buffer ( appendix 22)
  • Buffered phenol (unit 2.1)
  • Diethyl ether
  • 7.5 M ammonium acetate
  • 95% and 70% ethanol, ice‐cold
  • 10× T4 DNA ligase buffer (unit 3.4) containing 5 mM ATP
  • 1 µg/µl phosphorylated EcoRI linkers, 8‐ or 10‐mers ( Collaborative Research)
  • T4 DNA ligase (measured in cohesive‐end units; New England Biolabs; unit 3.14)
  • EcoRI restriction endonuclease and 10× buffer (unit 3.1)
  • 10× loading buffer without Xylene Cyanol (unit 2.5)
  • recipeCL‐4B column buffer
  • Agarose, electrophoresis‐grade
  • TBE electrophoresis buffer( appendix 3A)
  • Ethidium bromide solution (unit 2.5)
  • DNA molecular weight markers (unit 2.5)
  • 10 mg/ml tRNA
  • 3 M sodium acetate
  • 65°C water bath
  • 5‐ml CL‐4B column (see protocol 3)
  • Additional reagents and equipment for cDNA synthesis (unit 5.5), quantitation of DNA ( appendix 3A), agarose gel electrophoresis (unit 2.5), and fragment purification (unit 2.6)
NOTE: See discussion of preparation of buffer stocks in reagents and solutions section.

Alternate Protocol 1: Ligation of BstXI Synthetic Adapters

  Additional Materials
  • BstXI adapters (unit 2.11; Invitrogen), EcoRI adapters ( New England Biolabs), orEcoRI‐NotI adapters ( Invitrogen)

Support Protocol 1: Preparation of a CL‐4B Column

  Additional Materials
  • Preswollen Sepharose CL‐4B ( Pharmacia), 4°C
  • CL‐4B column buffer
  • recipeSilanized glass wool
  • Plastic tubing (new) with clamp
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Figures

Videos

Literature Cited

Literature Cited
   Hedrick, S.M., Cohen, D.I., Nielsen, E.A., and Davis, M.M. 1984. Isolation of cDNA clones encoding T cell‐specific membrane‐associated proteins. Nature (Lond.) 308:149‐153.
   Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Morgenstern, J.P. and Land, H. 1990. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper‐free packaging cell line. Nucl. Acids Res. 18:3587‐3596.
   Seed, B. 1987. An LFA‐3 cDNA encodes a phospholipid‐linked membrane protein homologous to its receptor CD2. Nature (Lond.) 329:840‐842.
Key References
   Neve, R.L., Hanis, P., Kosik, K.S., Kurnit, D.M., and Donlon, T.A. 1986. Identification of the gene for the human microtubule‐associated protein tau and chromosomal localization of the genes for tau and microtubule‐associated protein 2. Brain Res. 387:271‐80.
   Seed, B. 1987. See above.
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