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EcoRI site at the 5' end of the coding strand and a BamHI site at the 5' end of
the complementary strand. This allows the insertion of the cDNA into the
multiple cloning site of pLXSN in the "sense" orientation. If BamHI or
EcoRI cut within the insert, some of the other restriction sites may be
used. Before further use in transfection studies, the quality of the PCR product should be assessed by DNA sequencing (see Chapter 13).
Subprotocol 2
Purification
Procedure
DNA purification methods are described in Chapter 3. The vector DNA and
the PCR product should be cut with the appropriate restriction enzymes and
gel-purified. The DNA may be transferred onto a DEAE-cellulose membrane and eluted, or excised from the agarase gel and electroeluted. The
amount of DNA recovered from the final phenol/chloroform extraction
and ethanol precipitation must be determined (see Chapter 3 for DNA
quantitation).
Subprotocol 3
Ligation of the Insert into the Vector
Materials
• Gel-purified vector and insert, both digested with appropriate restriction
enzymes
• Ultra pure H20
• Ligase
• 10 x ligase buffer
• Sterile microcentrifuge tubes