TILLING Open Laboratory
What is TILLING?
NBRP-Rice opens TILLING Open Laboratory to select target-gene mutants in rice.
[Outline of the entire process]
We require that preliminary experiments for selection of target-gene mutant using TILLING be conducted at your laboratory. When you have completed your preliminary experiments, please make arrangements for scheduling, and then visit Faculty of Agriculture at Kyushu University for the selection of target-gene mutants.
First of all, please contact Dr. Kumamaru (kumamaruagr.kyushu-u.ac.jp).
Preliminary experiments are conducted to choose the most appropriate primer combinations and annealing temperature for TILLING. Because these conditions are very important, you should not make any compromises at this stage.
The following are cautionary guidelines for primer design.
- Please design primers about 100 bp upstream and downstream of the target sequence so that amplified products are under 1.5 kb. This is important because it is difficult to detect fragments shorter than 100 bp produced by CEL1 treatment in the subsequent electrophoresis step.
- Please determine annealing temperatures that produce only amplified fragments of the expected size using the primers you have designed. Although these conditions are also examined using equipment in our laboratory, this process goes more smoothly if the conditions are also examined during preliminary work. This is important because it is difficult to distinguish non-specific bands from CEL1 fragments. In addition, please avoid using primers with poor amplification efficiency.
- Currently, Kinmaze, Taichung 65, and Nipponbare are used as the original varieties for the generation of mutant strains. Please conduct preliminary experiments using genomic DNA from these three varieties, and confirm that amplified fragments of the expected size are obtained from all three varieties.
- Please see the separate sheet for more details about selection of the polymerase.
[Term of selection]
The length of time required for selecting mutants depends on the number of primer combinations, but not the number of genes to be screened. As a rough guide, it takes 2–3 days to become experienced at pipetting for TILLING as well as to perform re-examination of conditions that were examined in the preliminary experiments, and another 2–3 days for selection for each combination of primers.
Currently, we preparing four plates of genomic DNA (template), dispensed in 384-well plates by strains. The number of strains selected with a single set of primer combination is about 1,500 strains. The price for screening of one plate, including all necessary steps (PCR, CEL1 treatment, and electrophoresis) is \ 6,170. Polymerase is not included in this price, so please bring your own polymerase. About two plates are used for re-examination of conditions, and this re-examination is charged at the same rate. We are currently preparing additional DNA, and the number of strains may increase.
Meanwhile, DNA plates (384-well) including CEL1 enzyme are also available for distribution. The price for one plate is \6,170, the same as the price for screening one plate.
For strains containing SNPs, we will provide the seeds (and DNA if possible) with the clients, who would then conduct genomic DNA sequencing. For provision of the seeds, separate fees are required in accordance with the billing rules of NBRP, Kyushu University. Furthermore, acquiescence to the Material Transfer Agreement (MTA) is required.
Mutant strains derived by TILLING are NBRP strains. Therefore, the obtained results should be handled as though they were from NBRP. We would like to ask you to publish the data on Oryzabase after acceptance of papers or presentations at academic conferences. We will ask your formal permission for publication at that time. Data samples are available here.
Kyushu University Guest House is available (\3,500–4,000 per night, meals not included); however, the rooms are limited in number. If rooms are not available at the Guest House, we will offer referrals to neighborhood hotels. If you would like to use the Guest House, please let us know so that we can check and adjust the schedule.
Please see here for more details (Japanese only) on the Guest House.
[The number of primer combinations]
When you contact us for the first time, please let us know the approximate number of primer combinations you will be using.
[Contact information for application]
Plant Genetics Laboratory, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University
Toshihiro Kumamaru (kumamaruagr.kyushu-u.ac.jp)
Web site: http://www.agr.kyushu-u.ac.jp/lab/pgr/
A map of the campus is available here. The laboratory is located in Building #75 (Faculty of Agriculture) in the science district.