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Production of a Subtracted cDNA Library

Lloyd B. Klickstein¹

¹Brigham and Women's Hospital, Boston, Massachusetts

Publication Name:

Current Protocols in Molecular Biology

Unit Number:

Unit 25B.1

DOI:

10.1002/0471142727.mb25b01s55

Online Posting Date:

August, 2001

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Abstract

For some experiments, a complete cDNA library is unnecessary and instead, a subtracted cDNA library is useful. A subtracted cDNA library contains cDNA clones corresponding to mRNAs present in one cell or tissue type and not present in a second type. This cDNA library is used to isolate a set of cDNA clones corresponding to a class of mRNAs, or to aid in the isolation of a cDNA clone corresponding to a particular mRNA where the screening procedure for the cDNA clone is laborious because a specific DNA or antibody probe is unavailable. Since relatively few recombinants are obtained after subtraction, this protocol is for a cDNA library constructed in the >10 vector or its equivalent, which allows a high cloning efficiency and permits elimination of nonrecombinants; however, the protocol can be used to produce subtracted cDNA libraries in any vector system.

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Basic Protocol: Production of a Subtracted Library
Reagents and Solutions
Commentary
Literature Cited

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Materials

Basic Protocol: Production of a Subtracted Library

Materials

[+] and [–] cDNA libraries (ATCC or Stratagene)
TE buffer (APPENDIX 2)
EcoRI and 10× EcoRI buffer (UNIT 3.1)
0.5 M EDTA, pH 8.0 (APPENDIX 2)
10% sucrose solution (UNIT 5.3)
1.5% and 2% agarose gels (UNIT 2.5A)
TBE buffer (APPENDIX 2)
95% and 70% ethanol
S1 nuclease (Sigma; UNIT 3.12) and 10× S1 nuclease buffer (UNIT 3.4)
25:24:1 phenol/chloroform/isoamyl alcohol (UNIT 2.1A)
3 M sodium acetate, pH 5.2 (APPENDIX 2)
AluI and 10× AluI buffer (UNIT 3.1)
RsaI (UNIT 3.1)
Deionized formamide (Fluka, IBI, or American Bioanalytical)
20× SSC (APPENDIX 2)
1 M NaPO₄, pH 7.0 (see recipe)
10% sodium dodecyl sulfate (SDS)
10 mg/ml yeast tRNA
24:1 chloroform/isoamyl alcohol
Phosphatased gt10 arms (Stratagene)
10× T4 DNA ligase buffer (UNIT 3.4)
T4 DNA ligase (measured in cohesive-end units; New England Biolabs; UNIT 3.14)
E. coliC600hflA (Table 1.4.5)
phage packaging extracts (Stratagene)
Suspension medium (SM; UNIT 1.11)

SW-28 rotor and 38-ml centrifuge tubes (Beckman) or equivalent
0.4-ml microcentrifuge tube

Additional reagents and equipment for construction of recombinant DNA libraries (UNITS 5.5 & 5.6), large-scale DNA preps from plasmids (UNIT 1.7) or phage (UNIT 1.13), sucrose gradients (UNIT 5.3), agarose gel electrophoresis (UNIT 2.5A), production and growth/maintenance of phage libraries (UNITS 5.8A, 25B.2, and 1.9-1.3), plating and titering libraries (UNITS 6.1 & 6.2), hybridization (UNIT 6.3), and radiolabeling probes (UNIT 3.4)

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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.
	Lamar, E.E. and Palmer, E. 1984. Y-encoded, species-specific DNA in mice: Evidence that the Y chromosome exists in two polymorphic forms in inbred strains. Cell 37:171-177.
	Tedder, T.F., Strueli, M., Schlossman, S.F., and Saito, H. 1988. Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 85:208-212.