Table 2 PCR cycling conditions and primer information used for double stranded cDNA generation and colony PCR analysis, as described in SMART cDNA Library Construction Kit [18].
Process | Cycling Conditions | Primer information | |
|---|---|---|---|
cDNA synthesis | 95°C - 2 min ↓ 95°C - 30 sec 65°C - 30 sec 68°C - 4 min repeated 18-24 cycles ↓ 68°C - 15 min Hold at 4°C | SMART IV™ Oligonucleotide (10 μM) 5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3' CDS III/3' PCR Primer (10 μM) 5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N-3' 5' PCR Primer (10 μM) 5'AAGCAGTGGTATCAACG CAGAGT-3' | |
Colony PCR | 95°C - 5 min ↓ 95°C - 30 sec 55°C - 30 sec 72°C - 3 min repeated 29 cycles ↓ 72°C - 5 min Hold at 4°C | Forward Primer: Gal 1 5'-AATATACCTCTATACTTTAACGTC-3' Reverse Primer: V5/6XHIS 5'-ACCGAGGAGAGGGTTAGGGAT-3' | |