directional cDNA cloning

Mayumi Yagi myagi at u.washington.edu
Thu Jan 4 14:40:18 EST 1996


Hi, all,

I'd like some advice on directional cDNA cloning.  In the past I've 
successfully constructed nondirectional cDNA libraries, but for a variety 
of reasons we're considering doing a directional library in a homemade 
vector with EcoRI (5') and XhoI (3') sites.  Stratagene sells a kit using 
precisely these enzymes, but before popping $600 I'd like to get some 
feedback both on the kit and the potential difficulties I might encounter 
in the protocol. Essentially, the protocol substitutes methyldC for dC in 
the cDNA synthesis reaction, the idea being to prevent cleavage by XhoI 
at any internal sites.

I'd appreciate hearing from anyone who's used this kit, and any 
suggestions from others who have done directional cloning.

Thanks in advance,

Mayumi Yagi



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