Designing nested primer sets for LM-PCR (dsg 10/9/06)
Retrieve sequence for the region of interest from flybase and paste into a
text file.
Go to primer
3 to identify PCR primers. Paste sequence corresponding to
the region approximately 230 base pairs from the farthest region of
interest. For example, if Pol II initiates transcription at +1,
analyze the sequence from +1 to +230.
Focus the primer selection on the distal regions of the sequence.
Insert a "<" about 50 nucleotides from the 5' end and a ">" about
50 nucleotides from the 3' end.
Try identifying a nested set of primers with Tm's of 54oC, 58oC
and 62oC (+/-1oC). If this doesn't work,
try Tm's of 58oC, 61oC, 65oC (+/-1oC).
After identifying the first primer, remove part of the 3' end of the
sequence to force the search for the next primer towards the 5'
end. Repeat this to identify the third primer. The nested
set of primers should have increasing Tm's and overlap so that each
primer blocks the 3' end of the preceding primer from annealing to the
DNA.
Next, go to the IDT
web site to make certain this set of primers is not prone to
forming hetero or homo dimers. Also check for compatibility
with linker primer A', which is GCGGTGATTTAAAAGATCTGAATTC.
Finally, BLAST the primer sequences against the Drosophila genome using
flybase.