Dr. S. Bhattacharya
sbhattac at crc.ac.uk
Wed Dec 2 06:20:13 EST 1992
I've been trying to do anchor PCR from a lambda gt11 library without success.
My main problem appears to be the appeareance of bands when using the gt11
5' or 3' anchoring oligo alone, and few, if any, that are produced by a
combination of internal and anchor oligo. Raising the temperature or adding
Perfect Match gets rid of all bands, not just the 'spurious' ones. I designed the
anchor primers using Eric Lander's PRIMER program to give a Tm of 55, as this
was the Tm of my (perfect, not degenerate) internal nucleotide sequence.
What are the successful strategies for anchor PCR? Obviously, as the library
is expensive, it is difficult to perform the reaction at many different
conditions to establish the optimal one. Has anyone designed oligos for
lambda gt11 anchors that do not produce spurious bands, and if so would they
please let me know. Are nested PCRs useful? Or should I just blot and probe
the bands and sequence all that light up? What oligo concentrations are
optimal? I have been using 500 ng of each oligo in a 50 ul reaction with
5 ul of a ~10^8 cfu/ul once amplified library.
I would appreciate any comments and help.
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