Uploaded Zymo DNA extraction protocol

_posts/20231208_zymo_DNA_miniprepplusKit_Protocol.md

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+---
+layout: post
+title: Adult Coral DNA Extraction using the Zymo Quick-DNA/RNA™ Miniprep Plus Kit
+date: '2023-12-08 14:00:00'
+categories: Protocols
+tags: [DNA, Protocol]
+---
+
+**This protocol is based off of the [base Zymo protocol](https://github.com/chloe-gilligan/Gilligan_Putnam_Lab_Notebook/blob/master/protocols/Zymo_quick-dna_miniprep_plus_kit.pdf), which was adjusted for Putnam Lab needs by [Zoe Dellaert](https://github.com/zdellaert/ZD_Putnam_Lab_Notebook/blob/master/_posts/2022-10-03-Protocols_Zymo_Quick_DNA_RNA_Miniprep_Plus.md)**
+
+**Goal: to extract *high-quality* gDNA  from adult tissue of diverse stony coral species.**
+
+## Materials and Equipment
+
+- Zymo Quick-DNA Miniprep Plus Kit [HERE](https://www.zymoresearch.com/collections/quick-dna-kits/products/quick-dna-miniprep) [Protocol Booklet](https://github.com/chloe-gilligan/Gilligan_Putnam_Lab_Notebook/blob/master/protocols/Zymo_quick-dna_miniprep_plus_kit.pdf)
+- Heating block capable of heating to 70ºC
+- Centrifuge and rotor capable of spinning at 15,000 rcf
+- Plastics: 5 1.5 mL microcentrifuge tubes per sample, 2 PCR tubes per sample, several 1000 uL, 200 uL, and 20 uL pipette tips per sample.
+
+## First time opening kit: Reagent Preparation
+
+1. Reconstitute the lyophilized (freeze-dried) Proteinase K as indicated on the vial prior to use. For 20mg Proteinase K, add 1040 uL Proteinase K Storage Buffer. Mix by vortexing. Check kit contents and instructions to confirm prep steps. Store tube at -20 ºC, no need to aliquot.
+
+## Sterilizing working area to maintain a clean environment
+
+Clean bench with clean paper towels (spray solution, wipe down) in the following order:
+
+  1. 10% bleach solution  
+  2. DI water  
+  3. 70% ethanol
+
+Clean pipettes, tip boxes, and the controls on the heating block and centrifuge by squirting 70% ethanol on a paper towel and wiping them down. Wipe down gloves with 70% ethanol and RNAse cleaner. Do not spray solutions directly on the equipment.
+
+## Sample preparation: Adult Fragment Tissue Sample in Zymo DNA/RNA Shield
+
+  This protocol utilizes coral clippings stored in 1mL of [Zymo DNA/RNA shield](https://www.zymoresearch.com/products/dna-rna-shield) and kept at -80 ºC. Thaw samples to room temperature on benchtop. On a clean work surface (see above), stand up the tubes in numerical order and photograph to record qualitative qualities such as buffer color and amount of tissue in tube.
+
+### Notes on sample preparation
+
+  This protocol will start by taking 400 uL of the DNA/RNA shield from the sample tube into a clean 1.5 mL tube and performing a Proteinase K digestion in a new tube. However, there are several adjustments that can be made at this step.
+
+- If the **DNA/RNA shield looks very dark** (ex: this occurs a lot with highly pigmented species such as ***Porites*** spp.), only take 150 or 100 uL of the DNA/RNA shield from the sample tube into the new tube and dilute with 150 uL or 200 uL of clean [DNA/RNA shield](https://www.zymoresearch.com/products/dna-rna-shield) (provided in kit). Proceed as directed.
+
+- If the extraction is not yielding enough DNA or RNA and the **DNA/RNA shield looks very light-colored**, it could be that the lytic properties of the DNA/RNA shield did not break up the tissue enough to yield enough DNA and RNA from the buffer alone. In this case, you could bead beat this sample using the protocols described by other lab members [Emma L. Strand](https://emmastrand.github.io/EmmaStrand_Notebook/Zymo-Duet-RNA-DNA-Extraction-Protocol/), [Dr. Kevin H. Wong](https://github.com/kevinhwong1/KevinHWong_Notebook/blob/master/_posts/2019-03-13-Zymo-DNA-RNA-Extract-P.astreoides-Genome.md), and [Kristina Terpis](https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2022-03-03-20220303-RNA-DNA-extractions-for-Maggie-Johnson.md). This bead beating step could be done by adding beads to the original sample tube, or by transferring an aliquot (300 uL) of the DNA/RNA shield from the sample tube along with a clipping of the tissue fragment into a new bead tube. Add 500-700 uL of clean DNA/RNA shield to make up 800 uL - 1 mL for bead-beating and bead beat for 1-2 minutes at maximum speed. Then, centrifuge briefly and take an aliquot of 300 uL from this homogenized mixture into a clean 1.5 mL tube. Proceed as directed.
+
+### Note: I have used this protocol for samples fixed sampled as follows:
+1. With sterilized forceps (10% bleach, DEPC water, 70% ethanol, air-dried), I removed one small (5mm x 5mm) fragment from the tube, in which most tubes had 5-6 small fragments of this size. I sterilized forceps between each sample.
+	- If samples in tube are large, use steralized clippers to break a small piece 	
+3. I briefly transferred this fragment to a clean 1.5 mL tube on ice or used a kimwipe to tap off any excess storage solution and then immediately transferred the fragment to a 1.5 mL screw-cap tube with 1000 uL DNA/RNA shield and 0.25 mL of 0.5mm glass beads.
+4. Bead beat for 1-4 minutes on the vortex at max speed. I started off with one minute for all samples but added a minute of bead-beating for samples if the liquid still looked very light-colored after one minute of homogenization.
+5. Briefly spin down and remove 400 uL of supernatant into a clean tube. 
+
+
+## Protocol Steps
+
+### Proteinase K Digestion  
+
+- On a clean work surface (see above), stand up the tubes in numerical order and photograph to record qualitative qualities such as buffer color and amount of tissue in tube.
+- Label a clean 1.5 mL microcentrifuge tube for each sample with sample number. This is a temporary tube, so you can use a shortened version of the sample number if desired. Make sure your labeling is clear. Ethanol is used in this protocol and can very easily rub off permanent marker writing on tubes.
+
+1. Invert all sample tubes 3 times to mix buffer well before pipetting out supernatant.
+2. Into a clean 1.5 mL microcentrifuge tube, transfer 400 uL of the supernatant from the sample tube (see notes above).
+    - Add: 20 µl Proteinase K to each sample tube.
+    - Invert 3x to mix or vortex 10-15 seconds.
+3. Incubate for **20 minutes** at room temperature.
+4. Place original sample tubes back in the freezer box (in -80 ºC) that they came from. Always keep these as backup.
+
+### Lysis and Column Transfer
+
+- Turn on heating block to 65 ºC and fill a 1.5 mL microcentrifuge tube with DNA Elution Buffer. You will need 50 uL for each sample, so aliquot 0.5 mL per 10 samples. Place this tube in the heating block so it will be at 65 ºC when you are ready to elute.
+- Label two clean 1.5 mL microcentrifuge tubes for each sample. 
+- Set up and label a yellow DNA (IIC-XL Collumn™) column in a collection tube for each sample.
+- Set up a waste beaker for liquid Zymo Kit waste. This will be disposed of in the waste disposal area in the collection container for Zymo Kit Liquid Waste.
+
+5. After the digestion, Invert/flick to mix tubes and centrifuge at 9,000 rcf for 3 minutes to pellet. Spin tubes with hinges facing out so pellet will form along the wall with the hinge.
+    - Note on centrifugation: Depending on the centrifuge settings, it will count down time from when you press start or from when it reaches the desired speed. Ours counts down when you press start, so we add 30s to each spin as written to account for the time to get up to speed. Here, I would spin for 3 mins, 30s.
+6. Transfer all supernatant to a new, clean 1.5 mL microcentrifuge tube. Be careful to not disturb pellet.
+    - Add: **420 uL** (should be a 1:1 ratio with supernatant volume) of **Genomic Binding Buffer**
+    - Mix these two solutions by pipetting up and down inside the tube or vortex 10-15 seconds.
+    - Transfer the entire volume (~840 uL) onto the labeled IIC-XL Collumn™ filter column (will trap **DNA, yellow** colored) in a collection tube. Switch tips for each sample.
+7. Centrifuge at 15,000 rcf for 30 seconds and **SAVE FLOW THROUGH** from this spin.
+    - Discard liquid (flow-through) in collection tube.
+
+### DNA Extraction
+
+- Label your final DNA tube (clean 1.5 mL microcentrifuge tube) with the full sample number, "gDNA", your initials, extraction date, and any other important information (like project, extraction specifications, etc.) on the side. The lid should say at least the full sample number and "gDNA".
+- Label PCR tubes (we usually use PCR strip tubes) for a small aliquot of your sample for QC. Make sure your tube says the sample number (or a shortened version) and "DNA".
+
+8. Add: **400 uL** of **DNA *Pre-Wash* Buffer**
+    - Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker *(every time: do not tap tube on side of waste beaker to minimize splash-back)*.
+9. Add: **700 uL** of **gDNA *Wash* Buffer**
+    - Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker.
+10. Add: **400 uL** of **gDNA *Wash* Buffer**
+    - Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker.
+11. Transfer to new collection tube and dry filter by centrifuging at 15,000 rcf for 2 minutes.
+12. Discard collection tube and transfer the yellow DNA column to the final, labeled DNA tube.
+    - Add: **50 uL** of warmed DNA Elution buffer **gently, directly onto the filter without touching the filter**. 
+    - Incubate for 5 minutes at room temperature.
+    - Centrifuge at 15,000 rcf for 30 seconds.
+14. Aliquot 10 uL of your DNA into your labeled PCR tube for QC. Keep on ice.
+15. Place final DNA tubes in freezer box in -20 ºC.
+    - Heating block can be turned off.
+
+
+## Quality Control (QC)
+
+These steps analyze the quantity and quality of the DNA extracted.
+
+### DNA Quantity  
+
+Follow Broad Range dsDNA and Qubit [protocol](https://zdellaert.github.io/ZD_Putnam_Lab_Notebook/Qubit-Protocol/) to analyze sample **quantity**. Read standards once and record values, read all samples twice.
+
+### DNA Quality  
+
+If DNA quantity is sufficient (typically >10 ng/µL) follow the PPP Agarose Gel [Protocol](https://zdellaert.github.io/ZD_Putnam_Lab_Notebook/Gel-Protocol/) to determine DNA quality. "Good" DNA should form a distinct band a the very top of the gel.
+
+## Gel protocol for both DNA Quality (From [Kristina Terpis](https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-10-08-20211008-RNA-DNA-extractions-from-E5-project.md))
+
+ - Modified from this [protocol](https://zdellaert.github.io/ZD_Putnam_Lab_Notebook/Gel-Protocol/)
+ - Set up small gel box and insert gel tray into the box with rubber gaskets touching the long side of the gel box. Make sure gaskets form a flush seal.
+ - Make a thin 1.5% gel:
+    - Add 0.75g of agarose and 50ml of 1x TAE ("New" container) to flask
+    - Microwave for 45 seconds and remove from microwave using heat-protective glove.
+ - Once cool enough to hold from the bottom of the flask, add 1 uL of gel green stain.
+ - Swirl and pour into gel mold with two 14-well combs.
+ - Once solidified (~30 minutes), cover with 1X TAE ("Used" container) as a running buffer
+ - To each of the samples in the QC strip tubes (after Qubiting you should have about ~9 uL left),
+    - Add 1uL of purple Tri-Track loading dye into each PCR tube, spin down to mix.
+ - Load 3 uL of the 1kb GeneRuler ladder into the first well of each row. Load the DNA samples on the top row following the ladder.
+ - Run the gel for 60 minutes at 60 volts.
+ - Image using UV gel imager covered with black cover and orange filter.
+ - Expectations
+    - ""Good" DNA should form a distinct band a the very top of the gel.