Background

Today I’m going to present a method of collecting plant tissue for DNA extraction. This method is intended for performing extractions in a 96-well format, using a tissue grinder and ball bearings for homogenization prior to extraction. Prior to graduate school, I had a job in which I performed low-throughput extractions, mostly using individual microcentrifuge tubes. However, in graduate school I worked on plant breeding projects that required genotyping hundreds of individuals. Clearly the single-tube method wouldn’t cut it anymore. Ultimately, the extracted DNA will be used in either 96-well or 384-well plates, so it’s easiest to collect tissue in this format to begin with.

In additional posts I’ll outline procedures for grinding the tissue samples and performing the extraction itself using a relatively low-cost method. However, before getting to that, I need to touch on an important topic: harvesting tissue for the extractions. If you’ve ever done plant DNA extractions, you’re probably wondering why I’m bothering with this topic. It’s pretty easy to sample tissue from plants – you just snip off some tissue, put it in a tube, freeze it for future use, and that’s that. There are the usual extra complexities if you will be performing RNA extractions, but here we will specifically be focusing on DNA.

Harvesting tissue might sound like a trivial step, but it’s important to talk about because it can cause a lot of problems if you start performing extractions on hundreds or thousands of samples. Grinding won’t work for tubes that contain too much tissue, meaning that you’ll either have to live without DNA for those samples, or else try to remove excess tissue, risking cross-contamination. Without further ado, on to the protocol (after a quick disclaimer):

Disclaimer

I present some catalog numbers for products below. I am not paid by these companies; these are products that I have successfully used in the past (sometimes after considerable trial and error). Other companies may market similar products that are just as effective.

Materials

• 96-cell seed germination trays
• 96-well format 1.2ml microtiter 8-tube strips with racks – sterile (Corning #4413) or non-sterile (Corning #4412)
• Caps for tubes above (Corning #4418)
• Clippers or scissors
• Paper towels or Kimwipes
• Ball bearing dispenser for 96-well plates (Spex SamplePrep #2100)
• 4mm Stainless Steel Ball Bearings (Spex SamplePrep #2150)

Note: The ball bearings listed above are autoclavable. If you don’t need sterile ball bearings for tissue grinding, a much cheaper alternative is to use BB’s for pellet guns.

Reagents

• Isopropyl or ethyl alcohol (preferably not denatured) for wiping down scissors/clippers between each sample

Lab Equipment

• (Optional but recommended) laboratory lyophilizer

Protocol

0. Yes, step zero. First, of course, you need to grow the plants in 96-well germination trays that mirror the plates that the tissue will eventually go into. I prefer to use potting soil, because it allows you to keep the seedlings alive for some time (more on that in step 4 below). Some people simply use cotton balls or other cheap germination media. With eudicot species I imagine you could wait until after the cotyledons unfurl to sample tissue. With wheat, I typically wait until seedlings hit the two-leaf stage:

1. Before touching scissors to plant, you need to set up and label the plates. The tubes come in strips of 8, so each strip corresponds to a column of a plate (tubes will usually come set up in this orientation in rack. Here’s a picture of the general kind of product we’re talking about (this is a picture of Simport T100 Biotubes – I haven’t personally tried this product but it looks promising).

 

So, to prepare the tubes for grinding, you need to:

1. Use the bearing dispenser to place bearing(s) into each tube – I usually use two bearings for some extra grinding heft.
2. Label each plate (the plastic rack holding the tubes) with whatever plate identification scheme you’re using.
3. Also label the strip tubes themselves – otherwise if they were to fall out of their racks, you would have no way of telling them apart. To unambiguously label each strip, you need to give it a plate identifier, column identifier, and some way to tell which end is the top row (row A), and which is the bottom row (row H). If I am working on a project with multiple plates, I will often give each column consecutive numbers across plates. So, for instance, the first plate will have columns 1-12, the second 13-24, and so on. I label both ends of each strip, so that, for instance, the top (row A) of column 2 looks like:

and the bottom (row H) looks like:

4. IMPORTANT! If you have an odd number of tube strips overall, you’ll need to add an extra dummy strip. This should be set up exactly like the other strips (i.e. with ball bearings), and you can label it “D” or “N”. If we have an odd number of tube strips, we will need this extra one in order to keep our plates balanced when the tubes eventually get centrifuged during the DNA extraction.

 

2. Now that the tubes are labeled and are holding the bearings, it’s time to take the tissue samples. The point here is to take a moderate amount of tissue, and break it into a few pieces. Most importantly, you should strive to take roughly the same amount of tissue for each sample. For monocots, this is relatively easy. Folks working with dicots might need to do a bit more work to get tissue pieces to easily fit into the tubes. For wheat, I just cut a piece of leaf the length of my pinky:

Then cut it in half, and cut each of those halves into halves:

Then just place the four leaf pieces into the tube, and move on to the next sample:

I wipe down the scissors/clippers with alchohol between each sample to try and minimize cross-contamination.

3. If you will be freezing the tissue, then you can place the caps onto each strip as you go. If you are able to lyophilize the tissue, then leave the caps off. You can then place the tubes in the lyophilizer for about 48 hours, giving you nice, paper-dry tissue. I then cap the tubes and store the lyophilized tissue in the fridge, though it should be able to safely stay at room temperature for a long time, if fridge space is tight.
4. It’s also a good idea to save extra tissue in case something goes wrong during an extraction. There are three ways to do this:
   • Keep the plants alive until extractions are completed (easiest, but requires potting mix and may be impractical if extractions won’t be performed for a while)
   • Collect two sets of tissue samples (you might have guessed… this takes twice the effort and materials compared to taking single samples)
   • Save extra seed of all lines (sometimes extra seed isn’t available, and you’ll waste a week or two waiting for seeds to germinate for any repeat extractions)

Epilogue

That’s really all there is to it. You now have nice, consistent tissue samples ready for DNA extraction.