TITLE: Leaf Punch DNA Extraction
AUTHORS: Alex Rajewski, Cecilia McGregor

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a quick (and very cheap) genomic DNA extraction protocol for fresh leaf or seed tissue in many plant species. It should be use preferably for extractions that do not require exceptional lengths of DNA, and is suitable for routine PCR, sanger sequencing, and restriction digests. The protocol is modified from Edwards </span><span style = "font-style:italic;">et al.</span><span style = "font-style:italic;font-style:italic;"> </span><span style = "font-style:italic;">Nucleic Acids Research</span><span> 19(6): 1349, and has been adapted since then by Zach King, Jonathan Serrano, and Cecilia McGregor. Typical yeilds of DNA are ~200ng/µL.</span></div></div>

[STEPS]
?. [Preparation]
Collect young leaf (the size of a penny) into a 2 mL Eppendorf tube. Store tubes at -80°C until extraction.
?. [Grind Tissue]
Snap freeze the tubes in a dewar of liquid nitrogen (LN2).
?. [Lysis]
Prepare 750µL of 60% Edwards buffer and 40% 5M NaCl for each tube.
[Edward's Buffer]
[5M NaCl]
?. [Lysis]
Centrifuge the tubes for 5 min at 13,000 rpm. During this spin, proceed to the next step.
[spin at 13,000rpm]
?. [Precipitation]
While the tube are spinning, add 1mL of 100% ethanol into a new tube. This is the tube you will store the samples in. Clearly label it with your initials, the sample ID and date on side, and the sample ID on the lid.
[100% ethanol]
?. [Precipitation]
Once the tubes are done spinning, transfer 500µL of the supernatant into the ethanol tube, and gently invert several times to mix.
During the transfer to not disturb the pellet at the bottom of the tube.During the inversion, you may see your DNA precipitate as a white-ish, wispy cloud that that looks like snot. This is good.
?. [Precipitation]
Centrifuge for 5 minutes at 13,000 rpm.
[spin at 13,000 rpm]
?. [Wash]
Add 300µL of 70% ethanol and centrifuge for 5’ @ 13,000 rpm.
[70% EtOH]
[spin at 13,000 rpm]
There is no need to resuspend the pellet.The extraction can pause here, just put the tubes at 4ºC.
?. [Wash]
Carefully pour-off ethanol. Turn the tubes upside down and blot as above (speeds drying of EtOH greatly).
?. [Wash]
Allow the plate to sit ~10 minutes at 60ºC in to evaporate ethanol.
Check the tubes to make sure all ethanol has evaporated by visual inspection or wafting before you proceed to the next step. You should not see or smell any ethanol. Pellets are clearly visible.
?. [Resuspend]
Resuspend the pellet in 200 µl of 1x TE buffer (or water). Rotate plate upside down gently several times to mix.
Optional, put tubes on a shaker at 200rpm for ~30 minutes to resuspend.We have noticed that this method tends to co-extract RNA, so spec/nanodrop concentrations of DNA might be artificially inflated by RNA. Addition of RNase A helps with this, but this extraction does not seem to effectively remove RNase.
?. [Grind Tissue]
Freeze a plastic tube wrack by pouring LN2 into it until the LN2 no longer evaporates.
?. [Grind Tissue]
Quickly remove an Eppendorf tube with the leaf tissue from the dewar, warm the tube hinge with your fingers to prevent it from breaking, open the tube, and place it in one of the LN2-containing wells. Make sure that no LN2 gets into the tube. Using a plastic pestle, grind the tissue to a fine powder, transferring the tube to a new well every ~10 seconds to prevent it from thawing. Close the tube, return it to the dewar, and process the next sample.
?. [Solution Preparation]
Make 500mL of a 1M Tris solution by dissolving 78.8g of Tris-HCl into 500mL of DI water, adjusting the pH to 8 with HCl, and autoclaving.
[Tris-HCl]
[conc. HCl]
[DI water]
This recipe has approximate volumes for HCl and water. Also, confirm that you are using Tris-HCl and not Tris-Base. Either is fine, but substitutions will change the mass of Tris needed and also alter the starting pH of the solution.
?. [Solution Preparation]
Make a 5M NaCl solution by dissolving 146g of NaCl in ~400mL of DI water, bringing to 500mL, and autoclaving.
[NaCL]
[DI water]
?. [Solution Preparation]
Make a 0.5M EDTA solution by dissolving 93.05g of EDTA in ~400mL of DI water. Adjust the pH to 8 by adding ~20g of NaOH. Bring to 500mL and autoclave.
[EDTA]
[NaOH]
[DI water]
The disodium salt of EDTA will not dissolve until the pH is near 8.
?. [Solution Preparation]
Make a 10% SDS solution by dissolving 50g of sodium dodecyl sulphate in ~400mL of DI water. Heat it to 65ºC to dissolve, and then bring to 500mL. There is no need to autoclave this solution.
[SDS]
[DI water]
?. [Solution Preparation]
Prepare the Edward's Buffer by adding the following compenents, bringing to 1L with DI water, and then autoclaving.
[1M Tris, pH 8]
[5M NaCl]
[0.5M EDTA]
[10% SDS]
[DI water]
?. [Lysis]
Add 750µL of this Edwards/NaCl mixture to each tube, vortex, and place at 60ºC for 30 minutes, inverting 2-3 times during the incubation to mix.
[Edwards/NaCl Solution]
[at 60ºC with occasional inversion]
?. [Precipitation]
Optional, allow the tubes to sit at -20ºC for at least 15 minutes. This step can go overnight but is normally a good place to break for lunch.
[optional incubation at -20ºC]
?. [Precipitation]
Carefully pour-off the ethanol. Turn the tube upside down for no more than 2 seconds, as pellets can be slippery, and blot with paper towels.