Cloning PCR fragments

Bruce Elder elder at CGL.UCSF.EDU
Wed Jan 20 12:21:47 EST 1993


In response to my response to Yin Chai's questions about PCR cloning I have
received many inquiries about why pDK101 is so special a vector. I don't really 
know that it is, however we have had great success using it to clone PCR
fragments from 200bp to just over 3kb. I think the main advantage over
"store-bought" T vectors (PCR1000, etc.) is that pDK101 is a plasmid that
we digest to produce the T overhangs. Store-bought vectors of this type are
already digested and have a limited shelf life as does digested pDK101. We cut
about a ug and run it out in low melt agarose, isolate the band and store it at
4 oC until needed. We then melt at 65oC and add an aliquot to the ligation
(about 2 ul when I do it). The cut vector keeps for about 3-4 months, but this
is somewhat variable.

Bruce Elder
elder at cgl.ucsf.edu



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