Although the Matrix Mill manual provides a basic protocol for DNA extraction, we needed to optimize the protocol to increase its reliability with maize leaf samples. The following procedure was developed for seedling leaves, based on our tests, the manualís protocol, and the work of Paris and Carter (Plant Mol Biol Rep 18:357-360, 2000).
Protocol: 1. Collect fresh seedling leaf samples using a paper punch and forceps. Place each leaf disc flat into the well of a 96-well microtiter plate containing 50 mL of sterile water.
2. Place a stainless steel dowel into each well.
3. Cover the 96-well plate with Parafilm and place at -80 C for 5 minutes.
4. Transfer the Parafilm-covered plate to the Matrix Mill; use a strip of Parafilm backing paper to prevent the Parafilm from sticking to the top grinding plate.
5. As soon as samples have thawed, grind for 2 cycles (approximately 4 minutes).
6. Aliquot 50 L of extraction buffer (500 mM NaOH) into each sample well.
7. Grind for an additional 20 seconds.
8. Transfer 5 L of the ground extract from each well into 200 L of neutralizing solution (80 mM Tris-HCl pH 7.5; 1 mM EDTA) that was pre-aliquotted into a 96-well PCR plate. Mix well.
9. Cover the plate with Parafilm and store at -20 C.
10. We use 5 L of each DNA sample in a 20 L PCR reaction; we run half of this reaction (10 L) on a gel.
Notes: Sampling: A standard hand-held paper punch works well to collect uniform samples of approximately 4 mg, which fit exactly into the bottom of the well. Broad-tipped forceps are useful for transferring the sample into the well and can be used to flatten the leaf disc. Samples can be extracted immediately or stored in the plate at -20 C for several weeks prior to step 2. We make a "grid map" diagramming the placement of samples in each microtiter plate during collection, and keep the same map with each plate containing finished DNA preps. Although this protocol was optimized for seedlings, it has also been successfully used on adult leaf samples.
Grinding: The dowel pins often have a sharper end, and this end should be placed down against the flat sample. It takes a few minutes for the water in the frozen plate to thaw after placement on the Matrix Mill; you should hear the dowel pins move freely while cycling after the thaw. Samples should be checked between the first and second cycle, as samples will occasionally work their way up the side of the well. These can be readjusted with forceps for the final cycle. Care must be taken to avoid cross-contamination, as some of the extraction liquid can stick to the Parafilm; if this occurs, we use the other side of the Parafilm for the second cycle. Successful maceration produces a pale green fluid.
The addition of NaOH to the extraction (step 6) is not absolutely necessary, but it does improve the DNA preparation, based on a modest increase in PCR band intensity relative to a no NaOH control. However, we found that grinding the samples for extended periods (up to 5 minutes) after addition of NaOH did not affect sample performance, either positively or negatively, for PCR. Therefore, the 20 seconds of additional grinding is merely a convenient way to insure that the samples are well-mixed following addition of the NaOH.
Neutralizing: Our tests indicate that the final step in preparation of the DNA is the most critical for success. The highly basic extraction solution must be diluted appropriately in buffered Tris/EDTA. If too much of the extracted DNA solution is transferred, the PCR fails completely (Figure 1). With a 12-channel pipettor, all 96 samples can be diluted in about a minute. Debris at the bottom of each well should be avoided during transfer.
PCR and Storage: Diluted samples are ready for use as templates in PCR reactions. We often aliquot 5 ml samples into PCR tubes immediately following the extraction procedure, to avoid having to thaw the entire plate of DNA preps later on. Due to the ~5% failure rate of the procedure, it is important to have a positive internal control in each PCR reaction, to assess the quality of each sample. The extracted DNA can be stored at -20 C for a few months. We have observed decreased performance with 12-month old samples.
1. The relative volumes of Matrix Mill extract to neutralization buffer
are crucial to the success of PCR amplification. Template DNA for each
of the four samples was made for a different maize leaf. The following
dilutions (extract:neutralization buffer) were tested: 1) 2 ml: 200 ml;
2) 6 ml: 200 ml; 3) 10 ml: 200 ml; 4) 16 ml: 200 ml; 5) 20 ml: 200 ml;
6) 30 ml: 200 ml. Primers amplified as segment of the wild-type maize rop6
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