This method provides samples for PCR using 96-well titer plates and 12-channel pipettes, but it can also be used for samples in individual tubes. Our routine method for grinding tissue for DNA extraction involves freeze-drying followed by pulverizing with glass beads. While freeze-drying is preferred, this method has been used successfully without freeze-drying, see below. Specified equipment includes a Mini-Bead-Beater-8 to agitate tubes and plates, and a tool for loading glass beads into 96-well plates.
Tissues that are successfully and routinely processed for SSR-PCR mapping: developing endosperms and embryos; seedling leaf tissues; immature lateral branch buds and ear shoots; leaves from maturing and adult plants; punches from mature freeze-dried leaves.
Sample size: 0.1 grams fresh weight samples into 1.5ml tubes or 1ml deep 96-well plates. This is generally equivalent to 10-20mg dry weight. Mature maize leaves that were previously freeze-dried have also been used by collecting 20mg samples with a paper punch. When using 96-well plates, care to prevent cross-contamination of samples is necessary at this stage.
Freeze-dry: Samples in 96-well plates or tubes are first completely frozen (liquid nitrogen or ultracold freezer), and are then freeze-dried. The dried samples are stable and easy to store before processing and provide a more concentrated (DNA) sample than fresh tissue.
Grinding: Next, three 3mm glass beads per tube/well are added. To save time and headache a tool was built on campus to add beads to all 96-wells at one time (see below). The dried samples are pulverized by the beads. We use a Mini Bead-Beater-8 that has been modified by the company to hold 1 ml deep 96-well microtiter plates. Grinding takes only a few minutes for most tissues.
Extraction and preservation: The pulverized samples are then treated with ROSE solution at 90 C. The ROSE solution has SDS for lysis, a high concentration of EDTA to preserve the sample, and insoluble PVPP to bind and exclude inhibitory phenolic compounds. Before each use ROSE must be mixed to distribute the insoluble PVPP equally.
Rapid-One-Step-Extraction Solution (ROSE): 312.5mM EDTA; 10mM Tris, pH 8; 1% SDS; 1% insoluble polyvinyl-polypyrrolidone (PVPP) (w/v). Add 200uL ROSE solution to each sample and mix well to wet the powdered tissue samples. Then the samples are heated to 90 C in a water bath for 20 minutes with additional regular mixing. The samples may then be cooled rapidly at 4 C for 5 minutes to use immediately. The cooled samples can be stored cold or frozen, but we find that they are stable at laboratory temperature indefinitely.
Dilution: Dilution reduces the concentration of EDTA and SDS. Using wide bore pipette tips, we remove 3uL from the liquid portion of recently mixed samples, and dilute 200-fold into 600uL sterile water containing 1% PVPP. The diluted samples are thoroughly mixed and the PVPP is allowed to settle. PVPP can inhibit the PCR reaction. Diluted samples are less stable than the original crude concentrated ones.
PCR, agarose gel detection of SSR alleles: The method for PCR is described in detail at the MaizeDB website: ftp://ftp.agron.missouri.edu/pub/methods/ssr_methods.html. From the diluted sample, we use 2uL in a 15uL PCR reaction. Because primer sets do not always have exactly the same optimum annealing temperatures, we typically use 10 cycles with 1 C decremental annealing temperatures, from 65 C to 55 C, followed by 30 cycles at 55 C. ROSE treated samples diluted 200-fold have 0.2mM EDTA, which reduces the effective Mg2+ concentration, but does not interfere. To resolve PCR products we use 4.5% SFR-agarose (Amresco) gels made with 1X TBE and 0.27ug ethidium bromide per 150mL gel.
Additional Considerations: Samples are relatively insoluble. We always mix the samples thoroughly and allow a minute or longer for the bulk of tissue debris to settle. Routine mixing provides equivalent, uniform samples, especially when using 12-channel pipettes. When pipetting from crude samples in ROSE solution, we use pipette tips with a slightly larger diameter tip; or, cut the tip ends from standard (1-200ul) tips with scissors. Because the samples can sometimes clog the tips of standard narrow-bore pipette tips, this helps to reduce differences between samples. In addition, crude samples should not be centrifuged, because the bulk of the DNA is probably still associated with insoluble cellular debris.
Alternative method to freeze-drying: We have explored using cellulase treatment of fresh tissue as an alternative to freeze-drying and have had good results. For each sample, add (~50ul) 1% cellulase in pH 6.5 buffered solution to thoroughly wet 0.05-0.1g tissue, incubate at 37 C for 1-2 hours, then freeze. The ROSE solution is added directly to these samples. A higher concentration ROSE solution (391mM EDTA, 0.012mM Tris, 1.25% SDS, and 1.25 % PVPP) may be used. The samples can then be treated as described above, if briefly homogenized by hand with a small pestle.
Equipment sources: DynaBlock1000 96-wells, 1ml deep with cap-mat lids: Research Products International Corp., Mt. Prospect, IL 60056-2190, www.rpicorp.com
Lyophilizers: Labconco, Kansas City, Missouri 64132-2696, www.labconco.com
Mini-BeadBeater-8TM modified for 96-well plates (<$2000): BioSpec Products, Inc., Bartlesville, Oklahoma 74005-0788, www.biospec.com
3mm glass beads: Jaygo, Inc. Union, NJ 07083, www.jaygoinc.com
Bead counter to add beads to 96-well
plates ($550): University of Missouri, Science Instrument Machine Shop,
Columbia, MO 65211, www.research.missouri.edu/web_research/ internal_funding/res_facil/machines.html
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