The following a compilation of primer sets that we have used for LM-PCR.  They are in sets of 3 with the first primer being the one used first in the procedure.  The second column is the Tm calculated using Pedro's molecular biology tool.  The third column is the annealing temperature we have used successfully in the PCR reaction.  The locations of primers TR-1 to TR-6, primers Try-D, A, and B, and primers 70ter-1, 2, and 3 are relative to the strart of a -194 to +84 hsp70 promoter construct in Car20ZT.2.  The EndoA, B and C are relative to the start of an endogenous copy of hsp70 at 87C.

All of these primers are being used in combination with the same "linker pair" which is ligated to the end of the double stranded DNA after the first extension in the LM-PCR procedure.  This pair consists of: Linker A', GCGGTGATTTAAAAGATCTGAATTC and Linker B, GAATTCAGATC.
 
Primer (numbers are relative to transcription start)
Calculated Tm
applied annealing temp.
TR-1: ACGACGTTGTAAAACGAC (+143 to +126 in p70w)
51
50
TR-2: GTAAAACGACGGGATCGATTCC (+135 to +114)
64
58
TR-3: CGACGGGATCGATTCCAATAGGC (+129 to +107)
70
62
TR-4: CTTCTGGTGCCGGAAACCAG (+303 to +284 in p70w)
65
60
TR-5: GCCGGAAACCAGGCAAAGCG (+295 to +276)
71
65
TR-6: CCAGGCAAAGCGCCATTCGCC (+287 to +267)
75
68
Try-D: CGAAAATATAAGCTCAATC (-254 to -236 in p70w)
47
46
Try-A: GCTCAATCAAAAGAAGC (-243 to -227)
48
48
Try-B: CAAAAGAAGCTTGGCTGC (-236 to -219)
57
53
EndoA: CAGTAGTTGCAGTTGATTTAC (+155 to +135 in 87C version of hsp70)
50
53
EndoB: GCAGTTGATTTACTTGGTTGC (+147 to +127)
57
56
EndoC: GCAGTTGATTTACTTGGTTGCTGG (+147 to +124)
64
60
70ter-1: CGTCAGTATCGGCGGAATT (+3103 to +3121 in p70w)
61
56
70ter-2: TATCGGCGGAATTCCAGCTGA (+3109 to +3129)
67
62
70ter-3: GAATTCCAGCTGAGCGCCGGTC (+3117 to +3138)
72
68
70ter-4: TTAATAATTTGCGAGTACGCA (+3495 to +3475 in p70w)
56
53
70ter-5: GAGTACGCAAAGCTTGGGCT (+3483 to +3464)
62
58
70ter-6: AAAGCTTGGGCTGCAGGTCG (+3475 to +3456)
67
62
TRE-A: AAGTCGAGCTCGAGCG (-55 to -40 in TRE-70w)
 54
52
TRE-B: TCGAGCGCCGGAGTATAAATAG (-46 to -25 in TRE-70w)
 63
58
TRE-C: GCCGGAGTATAAATAGAGGCGCTTC (-40 to -16 in TRE-70w)
 66
62