50 µl reaction mix for LCR:

*5 µl of 10x T4 ligase buffer (NEB)
*5 µl of 10x Ampligase buffer (Epicentre Biotechnology)
*0.67 µM of TmPrime-optimized oligonucleotides for target gene (phosphorylated using 20 U of T4 polynucleotide kinase, NEB), and 20 U of Ampligase (Epicentre Biotechnology)
* H2O to 5 µl.

LCR:
37°C for 4 hrs, denatured at 95°C for 3 min, ramped to 60°C at 0.1°C/s for annealing, and incubated at 60°C for 2–8 hrs.

PCR mix:
*2 µl of LCR product
*0.4 µM of outer primers
*2 mM of MgSO4
*500 µM of each dNTP (Stratagene)
*500 µg/ml of bovine serum albumin
*1 U of KOD Hot Start (Novagen)
*1x PCR buffer (Novagen)
*H2O to 50 µl.

PCR:
–2 min of initial denaturation at 95°C
–30 cycles of:
*95°C for 5 sec
*55°C for 30 sec
*72°C for 30 sec
–Last Extension at 72°C for 10 min.