Contracting Primer Extension
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Mon Aug 31 06:13:13 EST 1998
David L. Haviland, PhD wrote:
> You can not perform primer extension analysis from cloned material.
> THE only thing you could use your cloned genomic fragment for would be the
> sequence ladder (using the *same* oligo used in the primer extension
> assay) as a means of counting up the number of bases and determine where
> the TIS site is.
Well, no. There is one other thing you could do with your cloned material, if
it is in one of the current cloning vectors with RNA polymerase promoters (T3,
T7 ...) And that might be good in your case: You can use it to trouble-shoot
your primer extension assay.
* do a in vitro transcription from the cloned gene
* perform your primer extension reaction
* see if it gave the correct transcription start site (see the vector map for
the known start site)
* trouble-shoot until it works, then try on your REAL sample. Parameters to
optimize could be the annealing temperature, buffer additives like DMSO,
glycerol or alike, and, of course, the primer itself. It might be necessary to
order a new one to set up an assay that works.
Hope that helps,
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