284
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
when cultured in an hfl+ host. Important considerations regarding Iambda
gt10 cDNA libraries are reviewed by Huynh et al. (Huynh, Young and Davis
1984).
Many of the additional features listed above have been developed to
overcome some of the limitations of Iambda gtlO:
• The original vector has an EcoRI site as its only cloning site. As discussed
previously, there are numerous circumstances where one would prefer to
have alternative or additional cloning sites.
• Subcloning inserts from Iambda gtlO can often be a frustrating task.
Newervectors such as Lambda ZAPII (Stratagene) or A.SHlox (Novagen)
have novel excision systems that allow direct rescue of the insert as a
plasmid.
• Many investigators seek to identify genesthat are differentially expressed
between two tissues or cell types. A variety of subtraction protocols have
been developed that allow enrichment for clones that differ in their Ievel
of expression. Typically, pools of sense and anti-sense single stranded
molecules derived from each of the libraries are required. V ectors
such as Lambda ZAPII and A.SHlox have multiple cloning sites that allow
unidirectional cloning (to preserve 5'- 3' orientation) and flanking RNA
promoter sequences. The pools are generated using RNA polymerases
that prime from the respective RNA promoter sequences that flank
the cloning sites.
Procedure
Preparation of cDNA inserts for unidirectional cloning - guidelines
1. First strand cDNA synthesis is performed usingXhol primer-adaptors, 5methyl-dCTP, dATP/dTTP/dGTP, and reverse transcriptase. This reaction yields single-stranded cDNA molecules that are methylated at all
deoxycytosines. Many investigators choose to simultaneously perform
a tracer reaction using an aliquot of the final mix which allows them
to determine the efficiency of the reaction. A small amount of
a[3 2 P]dCTP is added to a fraction ofthe total reaction, and the two samples are incubated for 60 minutes at 42°C. A portion of the labeled reaction is removed for gel electrophoresis, another sample is allowed to dry
to completion (to determine total number of counts in sample), and a
third fraction is used to determine incorporated counts (number of
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
when cultured in an hfl+ host. Important considerations regarding Iambda
gt10 cDNA libraries are reviewed by Huynh et al. (Huynh, Young and Davis
1984).
Many of the additional features listed above have been developed to
overcome some of the limitations of Iambda gtlO:
• The original vector has an EcoRI site as its only cloning site. As discussed
previously, there are numerous circumstances where one would prefer to
have alternative or additional cloning sites.
• Subcloning inserts from Iambda gtlO can often be a frustrating task.
Newervectors such as Lambda ZAPII (Stratagene) or A.SHlox (Novagen)
have novel excision systems that allow direct rescue of the insert as a
plasmid.
• Many investigators seek to identify genesthat are differentially expressed
between two tissues or cell types. A variety of subtraction protocols have
been developed that allow enrichment for clones that differ in their Ievel
of expression. Typically, pools of sense and anti-sense single stranded
molecules derived from each of the libraries are required. V ectors
such as Lambda ZAPII and A.SHlox have multiple cloning sites that allow
unidirectional cloning (to preserve 5'- 3' orientation) and flanking RNA
promoter sequences. The pools are generated using RNA polymerases
that prime from the respective RNA promoter sequences that flank
the cloning sites.
Procedure
Preparation of cDNA inserts for unidirectional cloning - guidelines
1. First strand cDNA synthesis is performed usingXhol primer-adaptors, 5methyl-dCTP, dATP/dTTP/dGTP, and reverse transcriptase. This reaction yields single-stranded cDNA molecules that are methylated at all
deoxycytosines. Many investigators choose to simultaneously perform
a tracer reaction using an aliquot of the final mix which allows them
to determine the efficiency of the reaction. A small amount of
a[3 2 P]dCTP is added to a fraction ofthe total reaction, and the two samples are incubated for 60 minutes at 42°C. A portion of the labeled reaction is removed for gel electrophoresis, another sample is allowed to dry
to completion (to determine total number of counts in sample), and a
third fraction is used to determine incorporated counts (number of