RH Mapping Protocol

Considerations

Researchers who would like to put their zebrafish genes on the Tübingen radiation hybrid map have several options:

• Come to Tübingen to learn about our procedures and map your genes as a guest at our facility.
• Send us your primers or sequences to be mapped. We are willing to do this only on the basis of a collaboration.
• Perform the PCR in your own lab and send us the raw data. This will generally be the most convenient approach. An online form is available for data submission. Your genes will appear on the map as anonymous markers, however, we request that you allow us to reveal their identity after you have published them.
• You can also calculate a map on your own using our raw data in SAMapper format.

PCR Protocol

Set up 10 µl reactions:

• 7.14 µl reaction mix (1 µl of 10 x PCR buffer, 0.02 µl each of 100 mM dATP, dCTP, dGTP and dTTP, 6.06 µl water). 10 x PCR buffer contains 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl₂ and 0.1 % (w/v) gelatin.
• 0.08 µl each of 20 µM forward and reverse primer.
• 0.2 µl of 5 U/µl Taq polymerase.
• 2.5 µl of 10 ng/µl radiation hybrid or control DNA. Double the concentration for duplicates of markers that have initially given only faint bands.
  
  DNA of the Goodfellow radiation hybrid panel, including positive (zebrafish) and negative (Wg3H) controls, is available from Research Genetics, Inc. All our PCR reactions are set up by a Biomek 2000 robotic workstation (Beckman) on 96-well microtiter plates which are kept refrigerated below 10 °C. DNA samples of the 94 zebrafish radiation hybrid lines and the controls are prediluted on a deep-well plate. Reaction mix is stored in 1.5 ml aliquots at -20 °C. Each aliquot is sufficient for two plates. Before distributing to the plates, the robot adds Taq polymerase, divides each aliquot in half, and adds the primers.

Cycling:

• Initial denaturing at 94 ºC for 2 min.
• 35 cycles of denaturing at 94 ºC for 30 sec, annealing at 60 ºC for 30 sec and extension at 73 ºC for 1 min. Use 2 min extension time for markers that give a PCR product of more than 800 bp. Adjust the annealing temperature if necessary.
• Final extension at 73 ºC for 5 min.
  
  We run 12 plates in parallel on TouchDown (Hybaid) and Primus 96 plus (MWG-Biotech) cyclers. Of the markers on our initial map, only unp63 was annealed at 55 °C, and all others at 60 °C.

Electrophoresis:

• Add 5 µl of 6 x loading buffer (with approximately 10 ng/µl of undigested plasmid DNA as a loading control) to each sample.
• Run at 200 V for 45 min in 1 x TBE buffer, on 2 % agarose gels.
  
  We use Qualex Gold (AGS) gels with 8 x 30 lanes each and apply samples with a multipipette. The lanes are half as wide as the distance between microtiter wells, so that PCR products from two plate rows are interspersed on the gel. For each marker, one lane with 80 ng of 100 bp Ladder (Pharmacia) is run as a size standard.

Scoring:

• Run each marker in duplicate. Score even very weak bands as positive if they are clearly distinguishable from the background.
• In case of any discordancy (i.e. a hybrid scored as positive on one plate and as negative on the other), re-score the bands. If the discordancy persists, run a third plate and use the RH types supported by two of the plates. Because some PCR reactions may fail to give scorable bands, this procedure may leave a small fraction of the RH types undecided.
  
  We capture images with NIH Image 1.61 (developed at the U.S. National Institutes of Health, available at http://rsb.info.nih.gov/nih-image/) on a Power Macintosh 8500/120 computer equipped with a Cohu video camera and a Scion LG digitizer board, using on-chip integration to increase sensitivity as described in the NIH Image manual. We have created NIH Image macros for capturing and semi-automatic scoring of the images, and FileMaker Pro 3.0 databases for scheduling PCR runs, cataloguing images, storing RH types and browsing mapping data. This software is currently very specific to our lab, but we expect to release a cleaned-up and properly documented version in the near future to help researchers who want to map genes on a large scale. For a small number of markers the scoring can easily be done manually.