User:Etchevers/Notebook/Conference notes/2009/03/26

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Notes for primer design for candidate gene sequencing

With Tania, choosing primers to test in lethal fetal pathology. First gene is 36 exons, designed 31 amplicons but must keep the following things in mind! Now need to redesign most of them. Also for another gene, 6 amplicons; total of 48 made yesterday and remaining 11 to redo.

1. Choose preferentially around 60°C Tm

2. Maximum comfortable length 600 bp

3. At least 50 bp flanking each for two reasons:

  • branch points for splice construction - in particular 5' to the exon.
  • sequencing always swallows up first nucleotides with blobs of dye and so forth.
  • Heather 05:25, 26 March 2009 (EDT):

Finally finished them all - mapping to sequence, highlighting, and making chart with suggested amplification temperatures and amplicon sizes.

  • Heather 10:10, 26 March 2009 (EDT):


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