TITLE: EASY AND INEXPENSIVE NUCLEIC ACID EXTRACTION PROTOCOL FOR RECALCITRANT SPECIES  
AUTHORS: Lilian Matallana, Yusuf Kurt, William Kohlway, John Frampton, Ross  Whetten

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Background</span><span>: Some of the major contributions to the rapid genetic improvement of commercial plant species and crops come through the identification of genetic markers linked to specific traits with a marketable value that have been difficult or impossible to identify and improve with conventional methods. Next-Generation Sequencing (NGS) is a powerful platform that has enabled the exploration of the genome in different plant species. The success of NGS is dependent on the quality of DNA or RNA extracted. Methods and commercial kits for extraction of intact nucleic acids for many model plant species are available but the need exists still for high-throughput methods suitable for most non-model plants, in particular, recalcitrant plant species. 
</span><span style = "font-weight:bold;">Findings</span><span>: </span></div><div class = "text-block">- Our group has developed a detailed method that allows the isolation of genomic DNA or total RNA from different tissues from different plant species that yield enough material suitable for high-throughput SNP genotyping, sequencing applications and traditional downstream genetic analysis. </div><div class = "text-block">- One single operator can extract nucleic acids from 192 samples in about 3.5 hours but the protocol is completely or partially-compatible with automation depending on available equipment.</div><div class = "text-block">- We provide tips and comments that will help to improve the quality of the isolated material and solutions for everyday laboratory problems that will save time and money. - Little variations in the protocol allow the application of the method to plant tissues from fresh, dry or frozen samples. </div><div class = "text-block">- The protocol is easy to set up in any molecular research laboratory, uses inexpensive reagents and handmade solutions that can be stored for several months, does not require the use of liquid nitrogen, and works best with 15 - 30 mg of fresh, frozen or dry plant material.</div><div class = "text-block">- DNA and RNA have been isolated from at least 30 species, including especially recalcitrant forest trees from angiosperm and gymnosperm families. DNA has been also extracted from stored (2 to 6 years) silica-dried samples of species of Pinaceae and Nymphaeaceae families.
</div><div class = "text-block"><span>- The protocol is a home-made adapatation of different published protocols that can be used in combination with different nucleic acids extraction kits. For reference consult: </span><a href="http://biodiversitygenomics.net/site/wp-content/uploads/2016/01/2006%20-%20Ivanova%20-%20An%20inexpensive%20automation-friendly%20protocol.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Ivanova, N.V.  et al. 2006</span></a><span>, </span><a href="https://link.springer.com/article/10.1007/s11105-008-0029-4" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Ivanova, N.V. et al 2008</span></a><span>, and </span><a href="https://onlinelibrary.wiley.com/doi/full/10.1111/j.1755-0998.2007.02074.x" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Whitlock R. et al. 2008</span></a><span>. 






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[STEPS]
?. [Prepare lysate]
This section provides guidelines and instructions to isolate DNA and RNA from different types of tissues and plant species. A general protocol is described but users need to read carefully for specific instructions to improve the quality and yield of the isolated genomic material. We have tested both lysis buffers for DNA and RNA interchangeably with similar results but we encourage the users to test the buffers for their own type of tissues and downstream analyses. We provide a table with the material that has been working best for us.  Tip: The protocol can be used for the extraction of DNA and RNA from tissues that have lots of starch, lipids and reserved material, like megagametophytes. We recommend using the protocol for RNA extraction for isolation of both DNA and RNA because this one has a shorter period of  incubation.
65 °C
?. [Prepare lysate]
After grinding, tissue can be stuck at the upper part of the tube forming a layer that will not allow the buffer to pass through. Spinning at  (thirty seconds) before removing the caps is enough to pull down all tissue. Open tubes carefully and clean your gloves with tissues and ethanol   while removing the strip caps or sealing mat. This will reduce cross-contamination between samples. Tip: toothpicks can be used to pull down the tissue instead of the centrifugation step. Use only one per sample. For RNA: spin samples at  (15 seconds).
Centrifuge: 1000 34, 30 s
Centrifuge: 1000 33, 15 s, 4 10
?. [Prepare lysate]
Add  Lysis buffer. For RNA (LBR): Place minitube plates with ground samples for 1-2 minutes on ice to thaw the sample right before adding the lysis buffer. Keep the LBR on ice before adding it to each sample. Tip: Do not proceed until the buffer in all tubes is liquid. For DNA (LBD): add prewarmed LBD at  . Close tubes with strip caps.
600 µl
60 °C
?. [Prepare lysate]
Homogenize the lysate by shaking plates in the Tissue Lyser and repeat these steps if the lysate does not look homogenous. For DNA: 4-6 minutes at 25 Hz and For RNA: 3 minutes at 25 Hz. Keep plates and adaptors   in case you need to repeat this step.  While plates are shaking, prepare a second set of 192 clean 1.1 ml tubes in two plates to transfer the clean lysate. For RNA: Add  of  sarkosyl to each tube ( sarkosyl/ lysate)) and keep plates at  .An alternative for DNA: this step can be extended if samples are too old (more than two years in silica gel ) or have a high content of phenols. After the addition of LBD, shake the plates for 5 minutes at 25 Hz, then incubate at   for  (30 minutes). Repeat this step 3X for a total incubation of 1.5 hours. Spin plates at  (10 minutes) and go to step 2.
on ice
10 µl
10 µl
1 ml
4 °C
65 °C
Centrifuge: 5000 33, 10 min, 4 10
?. [Prepare lysate]
Spin the plates at  (30 seconds) to settle out of the liquid. For RNA: place tubes on ice. Remove the caps from all tubes and be sure to clean your gloves between rows.
Centrifuge: 1000 34, 30 s
?. [Prepare lysate]
Transfer 400 - 600 ul of lysate and add fresh LBD or LBR  up to 1 ml to the new clean set of tubes (step 1.3). For RNA: tubes might contain  of  Sarkosyl.  Tip: this step can be adjusted depending on the type and nature of the tissue. Increasing or decreasing the amount of lysate and the dilution may help to improve the results.
10 µl
?. [Prepare lysate]
Incubate for 10 minutes at  , mix by slowly flipping the position of the plate by hand every 5 minutes. Cool on ice for about 10 minutes, then spin  (1 minute).
65 °C
Centrifuge: 5000 33, 1 min, 4 10
?. [Remove proteins and debris]
Add  of WB (wash buffer) to each sample. Spin plates for  (1 minute). Discard flow-through. Repeat this step 2X.
750 µl
?. [Remove proteins and debris]
Add  of PWB (Protein Wash Buffer) to each sample. Spin plates for  (1 minute). Discard flow-through. Repeat this step 2X. Tip: plates can be spun another two minutes until all liquid goes down. We do not recommend to spin the plates for more than three minutes.
600 µl
?. [Remove proteins and debris]
Spin plates 1-2 minutes. Tip: Entending the centrifugation time by another two minutes can help ensure all liquid goes down through the glass-fiber filter. We do not recommend to spin plates for more than 5 minutes in total.
?. [Remove proteins and debris]
In a new empty deep-well plate, combine  of  ethanol and  lysate from step 1.6. Mix with a multichannel pipette. An alternative for DNA: In a new set of 8-strip minitubes, combine  of lysate from step 1.3  and  chloroform:isoamyl alcohol (24:1), under the fume hood. Close tubes with strip caps. Invert plates for ~20X gently by hand, then spin  (10 minutes). Tip: we strongly recommend to use a gas mask even if the work is done under a fume hood. Chloroform:isoamyl alcohol needs to be poured into a glass reservoir or petri dish; plastic containers made of polystyrene will melt and should be avoided.
900 µl
600 µl
400 µl
600 µl
Centrifuge: 5000 33, 10 min, 4 10
?. [Remove proteins and debris]
Prepare 96-deep well plates, airpore tape sheets covers, PALL-plates, PCR plates and sealing foils/strip caps for PCR plates. Tip: Keep the same labels and marks used for the minitube plates. Minimize the spinning times help to keep the quality of the filters in the PALL plates and therefore improve the quality and DNA yield. If a small amount of liquid remains in some wells, empty into waste by flipping over quickly before moving on to the next wash addition. Usually this is an indication that original lysate needs to be more diluted.
?. [Elute DNA and RNA]
Place the PALL plate on top of a PCR plate. Tip: Label the plate properly and make sure the positions match between plates (e.g. position A1 on PALL plate matches position A1 on PALL-plate and PCR plate).
?. [Elute DNA and RNA]
Spin plates for 1 minute, then hold at room temperature (in the hood) for  (5 minutes). For RNA: Keep plates on ice while drying on the hood.
?. [Cleaning DNA and RNA]
After isolation, the optional DNase or RNase treatments were performed. The nucleic acid concentrations and purity were measured using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and integrity was analyzed on an Agilent 2100 Bioanalyzer with Pico chips (Agilent Technologies, Waldbronn, Germany). Users can evaluate if this step is required based on the downstream analysis needed. Tip: Add DNases or RNases only with filtered tips. RNases and DNases need to be handled very carefully to avoid contamination of the equipment and degradation of RNA or DNA during the extraction.
?. [Cleaning DNA and RNA]
For RNA: To clean and concentrate RNA samples we use the RNA Clean & Concentrator- 5 TM kit from Zymo Research. For DNA:  To clean and concentrate DNA samples a variety of kits and protocols can be used depending on downstream analysis and requirements.
?. [Elute DNA and RNA]
For DNA: Elute DNA with 20 - 40 uL AE buffer (at 50 C), over 5 minutes at room temperature. For RNA: Elute RNA with 20 - 40 uL molecular grade water (DNase and RNase-free), over 2 minutes keeping plates on ice. Spin plates for  (1 minute).
?. [Elute DNA and RNA]
Cover each plate with a sealing foil or with 8-strip caps for PCR plates. For RNA: plates can be stored at . For DNA: plates can be placed at   or at  for long storage.
-80 °C
-20 °C
-80 °C
?. [Remove proteins and debris]
Transfer  of lysate from step 2.1 to the PALL-plate. Cover each PALL- plate with an airpore tape sheet and let them sit at least 45 minutes in  freezer. Tip:  30 -60 minutes have given the best results for most of the tested samples, longer periods of time at  or  increased yield but decreased the quality of the genomic material. An alternative for DNA: Transfer 150 - 200 ul of the supernatant directly to a PALL-plate and add 500 - 600 ul cold  ethanol. Tip: Be careful at removing the top layer (clear supernatant). Touching lower layers will have a significant effect on the quality. Cover each PALL- plate with an airpore tape sheet and let them sit at least 45 minutes in  freezer. Tip:  30 -60 minutes have given the best results for most of the tested samples, longer periods of time at -20 or -80 C increased yield but decreased the quality of the genomic material.
600 µl
-20 °C
-20 °C
-80 °C
-20 °C