RAPD cloning

Sasha Kraev bckraev at aeolus.ethz.ch
Thu Nov 17 04:53:02 EST 1994


Several years ago we cloned many RAPD fragments from cotton genomic DNA,
using blunt-ended M13 ( at that time sold by Amersham ) and plating it
on SURE strain from Stratagene. Since many of these fragments turned out
to be repetitive sequences, the vector/strain combination was chosen with
the idea of getting reasonable clone stability plus easy sequencing. We
did encounter problems with sequencing, since RAPD clones contained inverted
repeats at the ends (the primer), which could completely block sequencing
reaction if one used the standard protocol with slow cooling to r.t. It
was eventually overcome by using Taq polymerase for sequencing. Hope this
helps.
Sasha



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