DNA sample preparation
Obtain DNA ESTs from Stock Center or obtain isolated clones. Prepare a plasmid miniprep.
Set up PCR reaction :
| Reagent | Volume (µl) |
| 10X PCR buffer | 10 |
| dNTPs (10 mM each) | 2 |
| Primer 1 (50 µM)1 | 2 |
| Primer 2 (50 µM)1 | 2 |
| Miniprep plasmid DNA (10 ng/µl) | 1 |
| Taq DNA polymerase (5U/µl) | 1 |
| H2O | 82 |
| Total | 100 |
Amplify targets using 35 rounds of PCR .(94°C, 2 min, 35X[94°C, 1min; 55°C, 1min 30sec; 72°C, 1min 30sec], 72°C, 2 min).
Purification: Use PCR product cleaning kit (e.g. Qiagen QIAquick PCR purification kit Cat. # 28104)
Dry eluate (Speed vac) and resuspend in 8 µl H2O (0.1-0.5 µg/ml DNA) O/N at 4°C.
Transfer 5 ml DNA to V-bottom 384-well plate (MJ Research Cat. # MSP-384), mix with 5 ml 2X spotting solution (6XSSC, 3M betaine), cover with tape to avoid evaporation and store at -20°C until arraying.
Notes:
1. Primers derived from EST-containing vectors (T7, M13 forward for ZipLox or T3, T7 for pBluescript).
2. Alternatively: Add 1/10 vol 3M Na-acetate pH 4.8 and 2.5 vol EtOH (-20°C). Precipitate DNA 20 min at -20°C. Spin 30 min, 14'000 rpm, 4°C. Wash pellet with 70% EtOH (-20°C). Spin 10 min, 14'000 rpm, 4°C. Air dry pellet.
