| Production protocol | A) PCR Amplification from EST Clones
1. Clones are grown overnight in 384-well plates in LB+Amp (37C)
2. Resultant clone cultures are used (directly) as "template" for 15uL PCR reactions via three "inoculations" of 1X PCR mix usnig a 384-well plate replicator
3. PCR reactions for each array element are always conducted in duplicate and subsequently recombined in the product purification step (below)
4. PCR Master Mix (1X, 15uL reaction):
10mM Tris-Cl, pH 9.2
25mM KCl
3.5mM MgCl2
0.3uL dNTP's (stock = 25mM each)
0.04 uL Taq
0.003 uL forward primer (stock = 1mM)
0.003 uL reverse primer (stock = 1mM)
5. Thermocycles:
Step1: 95C for 2min
Step2 (39 cycles): 94C for 20sec, then 56C for 20sec, then 72C for 1.5min
Step3: 72C for 10min
B) Purification of PCR Products
1. We use the Genesis RSP 200 Liquid Handler (TECAN Inc.) and 384-well PCR filter plates (Millipore, catalog #S384PCR10) for high-throughput purification
2. Load replicate 15uL PCR reactions into a single filter well
3. Vacuum for 5min
4. No wash step
5. Re-suspend in 30uL water and mix thoroughly
6. Transfer to clean 384-well plates for spotting arrays
7. Evaporate water via speed vac
8. Re-suspend PCR products in 12uL spotting buffer (3X SSC, 1.5M betaine) for spotting arrays
C) Spotting the Arrays
1. We use the MicroGrid Pro arrayer (BioRobotics), MicroSpot2500 pins, and GAPSII slides (Corning)
2. Temp = 65-70 degrees F
3. Relative Humidity = 35-45%
4. Dwell time = 1sec
5. Spots per visit = 27
6. Pin wash = 7sec
7. Pin dry = 10sec
D) Processing Spotted Arrays
1. UV cross link the spotted arrays via 300 mJoules
2. Bake the cross linked arrays for 2 hours at 80-90C
3. Wash for 2min in 0.2% SDS
4. Rinse slides well via dunks in MilliQ water
5. Dunk in 90% EtOH
6. Spin dry for 2min (500 rpm)
7. Store at room temperature in a clean dessicator |
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