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# System prepended metadata

title: Plant Genomic DNA Prep without Liquid Nitrogen
tags: [molecular biology (tutorial), ' ebook']

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tags: molecular biology (tutorial), ebook
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# Plant Genomic DNA Prep without Liquid Nitrogen

## Summary: 
Plant Genomic DNA preparation is an important skill for any consideration of the flora in any ecosystem. Part of the attraction of this analysis is that it can be done anywhere at anytime. However, liquid nitrogen may not be available anytime, anywhere. We consider here an application for the genomic preparation of plant tissue that does not require liquid nitrogen - a difficult prerequisite for any field studies. Instead we used Powerbeads to lyse the sample sufficiently and generate genomic DNA of good enough quality for Nanopore sequencing

### Protocol:
In this method, we will mash the tissue in the microcentrifuge tube with a plastic pestle with the [Promega Wizard® High Molecular Weight DNA Extraction (A2920)](https://www.promega.com/products/nucleic-acid-extraction/genomic-dna/high-molecular-weight-dna-extraction-kit/?catNum=A2920#protocols), use the beads to break the cells and then purify using the rest of the HMW kit
1.	Measure 40μg by first taring epi and adding until 0.08g on scale
2.	Add 1ml of Buffer A from the Wizard HMW kit and mash with epi pestle. Perform this process until the large parts are made essentially into a slurry
3.	Transfer crushed solution with plant parts using a cut tip 1ml pipette tip (or "genomic" tips) to 2 ml Powerbead Pro Tube (Cat. No. 19301)
4.	Vortex complex mixture for 1 minute using a VWR tabletop vortexer set at midpoint(this step can likely be optimized but at 1 minute for *Plagiochila montagnei)*
5.	Add 2ml Powerbeads Pro Tube with complex mixture to 65°C for 15 minutes 
6.	Cool the lysate to room temperature for 5 minutes. 
7.	Centrifuge Pro Tube for 1 minute at 13,000*g*.
8.	Transfer the supernatant using a cut 1ml tip leaving beads and plant debris. Split the sample to get about 0.5ml in each tube
9.	Add 3μl of RNase A Solution to each sample and mix by inverting the tube 5–7 times. Incubate the mixture at 37°C for 15 minutes.
10.	Add 20μl of Proteinase K Solution to each sample and mix the sample by inverting the tube 10 times. Incubate the mixture at 56°C for 15 minutes. Cool to room temperature for at least 5 minutes or chill on ice 1 minute. This step cleaves the histones releasing the chromatin from its packaging.
11. Centrifuge at 13,000g for 3 minutes at room temperature to pellet any insoluble material. Transfer the lysate to a clean 1.5ml microcentrifuge tube.
12. Add 200μl of Protein Precipitation Solution to the nuclear lysate. Using 1ml cut tips, mix the solution five times pipette pumping the sample: (a) draw the tube contents from the bottom of the tube, (b) expel the lysate rapidly down the side of the tube and (c ) repeating. Small protein clumps may be visible after mixing. Incubate on ice for 5 minutes.
13. Centrifuge at 13,000g for 5 minutes at room temperature. Slowly transfer supernatants using a cut tip to a clean 1.5ml microcentrifuge tubes If any unpelleted debris is visible, repeat the centrifugation step.
14. Add 600μl of room-temperature isopropanol. Mix by inverting the tube 10 times. You may see smalll threads of white:DNA
15. Centrifuge at 13,000g for 2 minutes at room temperature. The DNA may be visible as a small pellet.
12. Decant the supernatant and add 600μl of room temperature 70% ethanol to the DNA. Gently invert the tube severaltimes to wash the pellet and the sides of the microcentrifuge tube. Centrifuge again at 13,000g for 2 minutes at room temperature
14. Carefully decant the ethanol. After decanting, perform a quick spin  at 13,000g and pull ethanol from opposite side of pellet. Invert the tube on clean absorbent paper and air-dry the pellet for 10–15 minutes.
15. Add 100μl of DNA Rehydration Solution to the tube. Do not vortex because this will cause mechanical shearing and decrease average fragment size. Rehydrate the DNA by incubating the solution overnight at room temperature. 

 
[Promega Wizard® High Molecular Weight DNA Extraction (A2920)](https://www.promega.com/products/nucleic-acid-extraction/genomic-dna/high-molecular-weight-dna-extraction-kit/?catNum=A2920#protocols)

Product name: PowerBead Pro Tubes (2 ml) (50)
Cat. No. / ID: 19301
Price: 249 USD