Annealing of complementary s.s. DNA's

bajenkin at swansea.ac.uk bajenkin at swansea.ac.uk
Mon Feb 20 11:08:51 EST 1995


In article <amatos-1302951347370001 at bib7.bc.edu>, amatos at hermes.bc.edu (Stephen Amato) says:
>
>  I have recently ordered some complementary oligo's from BioServe
>Biotechnologies.  These oligo's have 5' overhangs and I wish to label the
>annealed products using Klenow and alpha radiolabelled deoxynucleotides. 
>I have been having some trouble labelling them and I want to make sure the
>single stranded precursors are actually annealed.  Does anyone know of a
>method or of a reference that I could look up that would contain a
>protocol for actually following the annealing, maybe by absorbance at
>260nm?  I suspect one of think my other reactants may not be O.K., but I
>just want to make sure that my oligo's are indeed annealed.
>
>amatos at hermes.bc.edu
I used a similar procedure to synthesize a mutant DNA molecule
i.e. I constructed two complementary primers (89 and 128 bp) having a
30 bp 3' overlap. I tried to 'fill' the overhangs by PCR, but this failed
However if I used this failed PCR as a template for a subsequent PCR
I obtained the correct sized product, containing the mutations intended
Therefore there must have been correctly annealed primers in the first PCR
I hope this is of someGareth



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