Subcloning Blues

tera tjstbc at
Thu Feb 8 13:46:52 EST 1996

You should suspect the oligos used for PCR - many oligos that are 
synthesized have mutations due to the chemistry used for oligo 
synthesis.  The best way to check for restriction sites in the PCR 
product is to subclone the PCR fragment into a TA cloning vector and 
sequence through the ends to check for the proper restriction sites.  
You may want to PAGE purify the oligos before using them for PCR - the 
PCR fragments resulting from PAGE purified oligos (as opposed to OPC 
purified) seem to be more "cloneable".

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