• Hybridization of High Density Arrayed BAC Nylon Filter Blots

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Protocol for hybridization of high density arrayed Bacterial Artificial Chromosome nylon filter blots with 100 PCR isolated Unigene cDNA inserts, pooled in a combined probe

Isolation and purification of cDNA inserts using polymerase chain reaction

1. Thaw LB/10% glycerol stock plates containing Unigene clones.
2. To a 96 well PCR, thin walled plate containing 99µl of sterile ddH₂O per well, add 1µl of Unigene culture taken from the bottom of the glycerol stock wells.
3. Run BOIL program on the MJ Research Inc. thermocycler. 100°C 5min
4. Immediately place PCR plate on ice.
5. Transfer 1µl of boiled culture diluent into the wells of a new 96 well PCR plate.
6. Aliquot 24µl of PCR reaction mixture per sample well.

           Mixture      (each sample)
   
           2.5µl        10X reaction buffer (Perkin Elmer)
   
           2.5µl        MgCl2 (25mM)
   
           2.0µl        dNTPs (2.5mM)
   
           1.0µl        vector primer 1 (5µM)
   
           1.0µl        vector primer 2 (5µM)
   
           0.5µl        AmpliTaq (Perkin Elmer)
   
          14.5µl        ddH2O
   
   

7. Run PCR thermocycles.

   	Cycles	Time	               No.
   
   	94°	3min	                1
   
   	94°(30sec)55°(30sec)72°(50sec) 30
   
   	72°	5min	                1
   
   	 4°	hold
   
   

8. Stop with 2.8µl loading buffer.
9. Add 10µl or more of PCR samples to each well of a 1% low melt Sea Plaque GTG agarose, 1XTAE gel. Include a molecular weight marker lane.
10. Run standard parameters depending on type gel box used.
11. Cut out PCR product insert bands and place in 1.8ml microcentrifuge tubes.
12. Incubate 2hrs in 0.5ml 1X Gelase buffer (Epicentre Technologies) to equilibrate.
13. Remove all liquid from microcentrifuge tube, leaving the gel band slice.
14. Incubate band slice tubes at 70°C, 20min.
15. Transfer directly into 45°C.
16. Equilibrate 10min.
17. Add 1µl of Gelase (1U/µl) enzyme.
18. Continue 45°C incubation 2hrs .
19. Add 1 volume 3M NaAOc.
20. Add 2.5 volumes 200 proof Ethanol.
21. Mix.
22. Incubate -20°C, 1hr .
23. Microcentrifuge 13,500 rpm, 30min, 4°C.
24. Decant/aspirate supe.
25. Wash 1X 0.5ml 70% cold ethanol.
26. Microcentrifuge 13,500 rpm, 10min, 4°C.
27. Decant supe.
28. Air dry pellet.
29. Resuspend pellet in 20µl TE.