Production of a subtracted cDNA library.
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 lambda>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.
Klickstein
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Brigham and Women's Hospital, Boston, Massachusetts, USA.
D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D015246:Deoxyribonuclease EcoRI; D004926:Escherichia coli; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D007202:Indicators and Reagents; D012333:RNA, Messenger / Q000235:genetics; D013048:Specimen Handling; D013382:Subtraction Technique
D018076:DNA, Complementary; D007202:Indicators and Reagents; D012333:RNA, Messenger; D015246:Deoxyribonuclease EcoRI