Primer Information

Melting Temperatures (T_m's) for UI ASF custom primers

• M13 Forward custom primer (22-mer)
  5' - CACGACGTTCTAAAACGACGGC - 3' T_m = 72.3 C

• M13 Reverse custom primer (29-mer)
  5' - ATAACAATTTCACACAGGAAACAGCTATG - 3' T_m = 71.9 C

• T7 custom primer (26-mer)
  5' - GAATTGTAATACGACTCACTATAGGG- 3' T_m = 69 C

• T3 custom primer (23-mer)
  5' - CTCGGAATTAACCCTCACTAAAG - 3' T_m = 70 C

NOTE: Using the above dye-labeled custom primers allows us to run seperate sequencing reactions containing different primers at the same time on the same thermocycler. It also allows us to use a higher annealing temperature (60 C) for sequencing through compressions and difficult to sequence templates.

Melting Temperatures (T_m's) for LI-COR Standard Primers

The annealing temperatures for cycle sequencing protocols may need to be adjusted depending on the T_m of the primer being used. A good place to start is 5 C below the T_m of its primer. The T_m's of LI-COR standard primers are:

• 4000-20A M13 Forward (-29)/lRD41 Dye-Labeled Primer,
  19-mer: 5' - CACGACGTTGTAAAACGAC - 3' T_m = 58.1 C
• 4000-21A M13 Reverse/IRD41 Dye-Labeled Primer,
  20-mer: 5' - GGATAACAATTTCACACAGG-3' T_m = 54.1 C
• 4000-22A Lambda gtll Forward/IRD41 Dye-Labeled Primer,
  24-mer: 5' - GGTGGCGACGACTCCTGGAGCCCG - 3' T_m = 85.4 C
• 4000-23A T3 Promoter/IRD41 Dye-Labeled Primer,
  18-mer: 5' - AATTAACCCTCACTAAAG - 3' T_m = 47.5 C
• 4000-24A T7 Promoter/IRD41 Dye-Labeled Primer,
  20-mer: 5' - TAATACGACTCACTATAGGG; - 3' T_m = 51.3 C
• 4000-25A SP6 Promoter/IRD41 Dye-Labeled Primer,
  19-mer: 5'-GATTTAGGTGACACTATAG - 3' T_m = 44.4 C
• 4000-26A M13 Forward (-38)/IRD41 Dye-Labeled Primer,
  19-mer: 5' - TTTCCCAGTCACGACGTTG - 3' T_m = 63.2 C
  

NOTE: 4000-28 Custom Sequencing primers have their own unique T_m which is indicated on the Sequence Analysis sheet as T_m thermodynamic.