Will I lose half my adaptor by gel purifying?
jpcd0 at mole.bio.cam.ac.uk
Thu Mar 16 19:55:43 EST 1995
Hi, I have been ligating adaptors onto the ends of genomic digests to
enable me to clone the fragments into a particular site in a lambda. After
ligating on my adaptors (dephosph'd) the smear the digest will not
religate again, telling me that my adaptors have ligated on.
But the adaptors will only be covalently linked to the genomic fragments
by one strand ie:-
Dephosphorylated adaptor anneals but is not linked on upper strand
lambda arm-G a-a-t-t-a-g-g-c-c-g-c-a-g-c-g-g-c-c-t-c-g-a
Phosphate provided by lambda arm allows covalent linking to adaptor
So when I gel purify (to size fractionate and remove excess adaptors) will
I be left with:-
BTW the Tm as calculated by oligo for the double stranded bit of the
adaptor is 52.1 degrees
Will the voltage pull off the unlinked piece? should I adapt after gel
purifying the range of size I need?
John Dixon Lab 44 (223) 334131
Wellcome/CRC Institute Fax 44 (223) 334134
United Kingdom e-m: jpcd0 at mole.bio.cam.ac.uk
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