Will I lose half my adaptor by gel purifying?

John Dixon 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
lambda arm-C-T-T-A-A-t-c-c-g-g-c-g-t-c-g-c-c-g-g
                    *
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:-

lambda arm-G
lambda arm-C-T-T-A-A-t-c-c-g-g-c-g-t-c-g-c-c-g-g

????

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?

TIA

Johnny D

-- 
John Dixon                     Lab 44 (223) 334131
Wellcome/CRC Institute         Fax 44 (223) 334134
Dept Genetics
Cambridge University    
United Kingdom       e-m: jpcd0 at mole.bio.cam.ac.uk



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