TITLE: Low-cost tissue collection and genomic DNA extraction for plants
AUTHORS: Rachel Howard-Till, Claudia Osorio, Bradley Till

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes low-cost methods for the collection and desiccation of plant tissues and subsequent extraction of genomic DNA.  It contains modifications of previously described methods found at </span><a href="https://link.springer.com/book/10.1007/978-3-319-16259-1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://link.springer.com/book/10.1007/978-3-319-16259-1</span></a><span> and </span><a href="https://link.springer.com/chapter/10.1007/978-3-319-45021-6_14" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://link.springer.com/chapter/10.1007/978-3-319-45021-6_14</span></a><span>.  We have tested this protocol with leaf tissue from flax, Lupinus luteus, Camelina sativa and Berberis darwinii.  DNA is suitable for routine molecular biology assays such as PCR and also for next generation sequencing.  </span></div></div>

[STEPS]
?. Collection and dessication of plant tissues
?. [Collection and desiccation of plant tissues]
Collect plant tissue and place in a sealed container with silica gel orange, or equivalent. For larger samples, collect tissue in 50 ml falcon tubes. For Berberis darwinii leaves, which are tough and spiny, approximately 30 leaves were placed in a 50 ml falcon tube, which was filled with silica. It is best to initially fill tubes partially, then shake to distribute the leaves in the silica. Then fill the remaining space with additional silica. For Berberis, tissue was incubated in silica at least 4 days  .
0 Room temperature
?. [Collection and desiccation of plant tissues]
For smaller amounts of tissue, especially small soft leaves, one can place tissue in eppendorf tubes, and either fill tubes with silica and cap, or place open tubes in a box set inside a sealable container, cover with a breathable material such as cheesecloth or disposable cleaning cloth, then layer the silica gel over the cloth (as pictured below - this avoids the time consuming task of removing silica from tubes). Soft tissue is generally dry after a 2 day incubation  .
0 Room temperature
?. [Grinding and lysis of dry tissue. ]
Grind dry tissue to a fine powder.
?. [Grinding and lysis of dry tissue. ]
For Berberis, grinding was performed on a Precellys Evolution tissue homogenizer (Bertin instruments, Montigny-le-Bretonneux, France) using modified Precellys lysing kit “Tissue grinding CKmix50_7ml.” Approximately 100 mg of dry tissue (7 leaves) was placed in a 7 ml screw cap tube with 6, 2.8 mm and 2, 5.0 mm ceramic beads. Tissue was ground with 3,   pulses at 4500 rpm, which produced a fine powder.
?. [Grinding and lysis of dry tissue. ]
To grind softer tissue in 2 ml screw cap tubes (for example L. luteus, Camelina sativa, or flax), use 3, 2.8 mm ceramic beads and grind once for 20s at 4500 rpm.
?. [Grinding and lysis of dry tissue. ]
Alternative grinding methods can be used, including manual grinding with a mortar and pestle, or in-tube grinding with sand, glass, or metal beads on a common lab vortexer (for example, see figure 4.1of https://link.springer.com/chapter/10.1007/978-3-319-16259-1_4).
?. [Grinding and lysis of dry tissue. ]
Lysis. Add RNase to LYSIS BUFFER (0.5% SDS, 10 mM EDTA, 2.5 µg/ml RNase).
?. [Grinding and lysis of dry tissue. ]
If grinding and lysis is performed in a 7 ml tube, as with Berberis, add  and shake or vortex tubes to completely suspend tissue. Shake tubes  on a multi-vortexer, or by hand.
[LYSIS buffer]
?. [Grinding and lysis of dry tissue. ]
If grinding/lysis is performed in 2 ml tubes, add  . Shake tubes  on a multi-vortexer, or by hand.
[LYSIS buffer]
?. [Grinding and lysis of dry tissue. ]
Incubate tubes  .
0 Room temperature
?. [Grinding and lysis of dry tissue. ]
Add   if lysing in 7 ml tubes, or  if lysing in 2 ml tubes, mix well. Incubate
[3M Na Acetate]
[3M Na Acetate]
on ice
?. [Grinding and lysis of dry tissue. ]
If grinding and lysis is performed in a 7 ml tube, distribute lysate slurry into 3  eppendorf tubes.
?. [Binding DNA]
Clear lysate and bind DNA to silica matrix.
?. [Binding DNA]
Clear lysate by centrifuging tubes from step 3 for 3 minutes at max speed in a microcentrifuge.
Centrifuge: 13000 33, 3 min
?. [Binding DNA]
Transfer lysate (~ 800 ul) to tubes containing BINDING BUFFER and SILICA (prepared in step 4.1). Flick or vortex tubes to resuspend silica. Incubate tubes  to at  , then  shaking on a multivortexer at  .
4 °C
0 Room temperature
?. [Wash and elute DNA]
Wash DNA bound silica pellets. Centrifuge  Discard supernatent.
Centrifuge: 13000 33, 3 min
?. [Wash and elute DNA]
Add  to tubes, flick or vortex to resuspend silica. Centrifuge  Discard supernatent. Repeat two more times for a total of three washes. After final wash, invert tubes briefly on paper towels and tap gently to remove as much liquid as possible. Then leave tubes open on their sides to dry pellets completely, approximately  .
[WASH buffer]
Centrifuge: 13000 33, 3 min
0 Room temperature
?. [Wash and elute DNA]
Elute DNA in TE with 12.5 ug/ml RNase A. Add   to the pellet, flick to resuspend. Incubate tubes  shaking at low speed.
[TE + RNase]
0 Room temperature
?. [Wash and elute DNA]
Pipette supernatent into a fresh 1.5 ml eppendorf tube.
Centrifuge: 13000 33, 3 min
?. [Wash and elute DNA]
For maximum yield, repeat elution. Combine the second eluant with the first.
?. [Binding DNA]
Prepare tubes with binding buffer and silica matrix. Ideally you can do this in advance, or during incubation steps above. Combine  (2 M guanidine thiocyanate in 67% EtOH - final concentrations) in a 2 ml eppendorf tube. Prepare one tube for each 2 ml lysis tube, or 3 for each 7 ml lysis tube.
[SILICA slurry]
[BINDING buffer]
?. [Collection and desiccation of plant tissues]
Check tissue before grinding.  Tissue should be brittle and break into pieces when pinched with fingers. If tissue is soft and bends, incubate with silica gel for a longer period of time. Note that silica gel orange and similar silica gels contain a color indicator.  If silica is saturated with water and color changes, replace with fresh silica. Silica gel can be dried and re-used. Tissue can be stored for many months to indefinitely at room temperature as long as silica is not saturated with water.
?. [Wash and elute DNA]
To ensure complete removal of RNA, incubate DNA  . DNA can then be concentrated by EtOH or PEG precipitation. We recommend PEG precipitation as it can help remove any protein impurities. We use a final concentration of 13.3% PEG 6000 and 10 mM MgCl2, with a   incubation at   followed by a    centrifugation.
4 °C
0 Room temperature
Centrifuge: 13000 33, 10 min
DNA stored in TE buffer can be stable for many years at four degrees Celsius. It is important to test the quality and quantity of your genomic DNA and the stability of the DNA in the chosen storage conditions.  For an example of how to do this, please refer to our protocols.io protocol describing quantification of DNA using a home-built low-cost gel documentation system.