Aug 01, 2023

Public workspaceEnvironmental DNA (eDNA) COI PCR Amplification and Gel Electrophoresis Protocol

DOI
dx.doi.org/10.17504/protocols.io.dm6gp3wpdvzp/v1
  • 1Stanford University
Meghan M. Shea
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DOI: dx.doi.org/10.17504/protocols.io.dm6gp3wpdvzp/v1
Protocol Citation: Meghan M. Shea, Alexandria B Boehm 2023. Environmental DNA (eDNA) COI PCR Amplification and Gel Electrophoresis Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3wpdvzp/v1
Manuscript citation:
TBD
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 18, 2023
Last Modified: August 01, 2023
Protocol Integer ID: 85185
Keywords: eDNA, environmental DNA, PCR, gel electrophoresis, amplification
Abstract
This is a protocol for amplifying DNA extracts via PCR using broad COI primers (Leray et al., 2013) and confirming successful amplification via gel electrophoresis.

This protocol originates with environmental DNA samples collected onto 0.22 µm capped Sterivex filters, e.g. through this sampling and filtration protocol:
Protocol
Coastal Environmental DNA Sampling & Gravity Filtration Protocol
NAME
Coastal Environmental DNA Sampling & Gravity Filtration Protocol
CREATED BY
Meghan M. Shea
Samples are then extracted, e.g. through this extracted protocol, before proceeding through the current amplification protocol:
Protocol
DNA Extraction Protocol from Sterivex Filters
NAME
DNA Extraction Protocol from Sterivex Filters
CREATED BY
Meghan M. Shea
Acknowledgements & Attributions:

The PCR protocol is adapted from Curd et al. (2019; see Appendix 6) and the detailed first PCR protocol developed by CALeDNA (by Teia Schweizer, Emily Curd, and Rachel Meyer).

The gel electrophoresis protocol is adapted from an Agarose Gel Electrophoresis & Gel Visualization Protocol from the Boehm Lab (by Blythe Layton and edited by Wiley Jennings, Eily Andruszkiewicz Allan, and Winnie Zambrana), a 16S rRNA gene V4 Amplicon Sequencing Protocol from the Nelson Lab (updated by Lauren Kennedy based on the Schloss Lab V4 Protocol) and the detailed Gel Electrophoresis protocol developed by CALeDNA (by Teia Schweizer, Emily Curd, and Rachel Meyer).

We are grateful to the authors and editors of all the protocols above for creating and maintaining such helpful resources.

Attachments
Icon for Sample PCR Calculation Spreadsheet.xlsx
Sample PCR Calculati...
11KB
Icon for IDT jgHCO2198 Primer Spec.pdf
IDT jgHCO2198 Primer...
538KB
Icon for IDT mICOIintF Primer Spec.pdf
IDT mICOIintF Primer...
528KB
Image Attribution
Meghan M. Shea
Materials
General Laboratory Equipment:

EquipmentSpecific Model Used
PCR HoodCBS Scientific Co. (P-036-202) PCR Hood
VortexVWR Mini Vortexer
Mini CentrifugeOnilab (D1008) Mini Centrifuge
PCR & microcentrifuge tube holdersVarious
1000 µL pipette with sterile tipsVarious
200 µL pipette with sterile tipsVarious
20 µL pipette with sterile tipsVarious
10 µL pipette with sterile tipsVarious
10% bleach solution in spray bottleNA
>70% ethanol solution in spray bottleNA
RNase Away solution in spray bottleNA
UV Light SourceBioRad Universal Hood II Gel Doc System
Gel Box with casting trayVarious
Power supply for gel boxVarious


Note
It's best to have designated pipettes for working with PCR products.


PCR Supplies:


MaterialAmount NeededSourceLinkApprox. Cost
Qiagen Multiplex PCR Kit12.5 µL/rxnQiagen (206143)https://www.qiagen.com/us/products/discovery-and-translational-research/pcr-qpcr-dpcr/pcr-enzymes-and-kits/end-point-pcr/qiagen-multiplex-pcr-kit315.87/kit
2 µL mICOIintF forward primer (2 µM) and 2 µL jgHCO2198 reverse primer (2 µM)2.5 µL of each/rxnIntegrated DNA Technologieshttps://www.idtdna.com/pages/products/custom-dna-rna/dna-oligos$52.1/one order
8-well tube strips1/every 8 rxnsFisher Scientific (AB20005)https://www.fishersci.com/shop/products/easystrip-plus-tube-strip-ultra-clear-caps/AB2005$295.37/250 strips
1.5/2 mL LoBind Eppendorf tubes (either size works; prefer 1.5 mL for storage)SeveralUSA Scientific (4043-1021/4043-1048)https://www.usascientific.com/dna-lobind-microcentrifuge-tubes/p/DNA-LB-Micro-Tubes$38.95/250 tubes



Note
IDT spec sheets for primers are attached to this protocol. Primers were ordered with the overhang specified by Georgia Genomics and Bioinformatics Core, where samples are ultimately sequenced. Core primer sequences used are:

mlCOIintF: GGWACWGGWTGAACWGTWTAYCCYCC jgHCO2198: TAIACYTCIGGRTGICCRAARAAYCA


Gel Electrophoresis Supplies:



MaterialAmount NeededSourceLinkApprox. Cost
Agarose1.9 g/gelVariableNAUnknown
1X TAE buffer (0.04 M Tris-acetate, 0.002 M EDTA)125 ml/gelVariableNAUnknown
GelRed Nucleic Acid Gel Stain 10,000X12.5 µl/gelBiotiumhttps://biotium.com/product/gelred-nucleic-acid-gel-stain/Unknown
6X gel loading dyeVariableVariableNAUnknown
100 base pair DNA ladder10 µl/gelVariableNAUnknown

Note
Working stock of 1X TAE buffer made from 980 mL DI water + 20 mL 50x TAE




Set-Up
Set-Up
Wipe down 10 µL pipette with 70% ethanol and RNase Away. UV for 10 minutes on each side
Wipe down a staging area near the PCR hood with 10% bleach, 70% ethanol, and RNase Away
Wipe down ice bin with 10% bleach, 70% ethanol, and RNase away, fill with ice, and remove DNA samples, PCR-grade water, Qiagen Multiplex Mix, and primers from the freezer to thaw. Place bin in staging area near PCR hood
Clean inside of PCR hood (including tube racks, etc.) with 10% bleach, 70% ethanol, and RNase Away
Wipe down PCR hood pipettes with 70% ethanol and RNase Away
Clean any items that need to go into the PCR hood and can be UVed with 10% bleach, 70% ethanol, and RNase Away and then place into PCR hood, including:
  • Lab marker
  • Bag of 8-well tube strips
  • Bag of microcentrifuge tubes
  • Strip tube holder
  • Any new tip boxes (make sure you have enough in the hood for the rest of the protocol)
With PCR hood cover in place, run UV light for 10 minutes
While PCR hood is UVing, wipe down a vortex, centrifuge, work bench, and box of 10 µL pipette tips with 10% bleach, 70% ethanol, and RNase Away
Once UV timer on PCR hood is done, remove the cover and turn on the regular light
Once reagents have thawed, gently vortex and wipe down the tubes with RNase Away before bringing into the hood
Place a Kimwipe dampened with RNase Away just outside the hood to clean tubes you bring in and out of the hood
Set up your diagram of how you're organizing your samples and calculate the total amount of PCR reagents needed for all samples, e.g. using the attached Sample PCR Calculation Spreadsheet

PCR Preparation
PCR Preparation
Change gloves before beginning to work in the hood
Label tube strips with relevant information, including:
  • Tube number
  • Initials
  • Date
Add primers and PCR-grade water to microcentrifuge tube(s) according to your combined reagent recipe (see attached Sample PCR Calculation Spreadsheet), remove from hood, vortex vigorously, wipe with RNase Away, and return to hood

Add Qiagen Multiplex Mix to microcentrifuge tube(s) according to your master mix recipe, remove from hood, vortex VERY gently, wipe with RNase Away, and return to hood
Note
Taq Polymerase does not like to be heavily vortexed

Using a pipette, aliquot 24 µL of master mix to each strip tube
Note
The amount, 24 µL, should be the final volume per reaction minus the amount of DNA template. You can reuse your tips unless you think you contaminated the tips.

Adding Template DNA
Adding Template DNA
Remove the aliquoted master mix and put on bench
Vortex each DNA sample and pipette 1 µL into the appropriate tube strip. Mix the DNA and master mix solution by pipetting up and down
Thermocycling
Thermocycling
Turn on thermocycler and select your program.

PCR cycling adapted from Curd et al. 2019
Note
Even once you've programmed your thermocycler settings, always check the program before using.

Place your samples in to the thermocycler, and double check that all tubes are fully sealed
The thermocycler will take between 2 and 4 hours to complete the program. While the thermocycler is running, prepare for gel electrophoresis (below)
When the thermocycler has finished running, remove your tubes and turn off thermocycler
Spin your PCR tubes down using a centrifuge or microcentrifuge specialized for plates or tubes and label. Make sure the labels are clear.
Note
If you will use your PCR products within 1 week, it is safe to store it at 4oC. If it will be more than 1 week, store your PCR products in a -20 oC freezer to avoid DNA degradation.

Gel Electrophoresis Preparation
Gel Electrophoresis Preparation
Weigh out 1.9 g of agarose and measure out 125 ml of 1X TAE for a 1.5% gel
Heat agarose in TAE in microwave until the agar is completely dissolved. You will know the agarose is completely dissolved when there are no specks floating in the TAE.
Note
Be careful of boil-over as it occurs quickly and unexpectedly. Recommend heating for ~30 seconds at a time and swirling in between.

Note
Use a paper towel or hot-pad to hold the beaker as it will be very hot.

Allow the solution to cool to just above room temperature. Placing the flask on a few folded paper towels will prevent the agarose on the bottom from solidifying too soon.
Once cool, add 12.5 ul of GelRed per 125 mL of gel (want it to be 1X final concentration). Gently swirl until GelRed is dissipated evenly.
Fit the casting tray in the box
Note
Make sure the casting tray is oriented so that the solution does not run off. Also make sure that the rubber gasket is seated properly so that the casting tray does not leak.

Pour the agarose into the casting tray and place the combs in the tray. Use the “fatter” side of the combs to make it easier to pipet into.
Allow the gel to solidify
Note
Gel is cool when it is a uniform milky white color. If the center looks clear still, it needs more time to solidify. It usually needs to cool for ~15-20 minutes.

Remove the tray and put in running position with the combs closer to the negative (black) electrode
Fill the box with TAE buffer until it just covers the gel
Carefully remove the combs
Note
At this point, you can store the gel for later if needed. Fill a shallow container (e.g,. Tupperware) with enough 1X TAE to cover the gel. Carefully slide the gel out of the casting tray into the TAE. Cover with tin foil and store at 4°C.

Decide the subset of samples to visualize via gel electrophoresis to confirm successful amplification
Gel Electrophoresis
Gel Electrophoresis
Load 5 µl of DNA ladder (at concentrations specified by the manufacturer) into the wells on either end of the row of samples
Dot 2 µl of loading dye (one dot per PCR product) onto a piece of parafilm
Note
If you have a large number of samples it can be easier to do this in a 96-well plate using a 12-channel pipetter.

Mix 4 µl of PCR product with the dye by pipetting up and down
Load the entire ~6 µl sample into a well, ejecting the sample slowly and carefully to avoid spillage or bubbles
Note
If there are any air bubbles in the wells, remove them with a pipet tip before loading sample into the well.

Note
Insert only the tip of the pipet tip into the well so you don’t pierce the bottom of the gel.

Run gel at 110 volts for 60 minutes
Note
These parameters can be adjusted for various applications, i.e. longer running times at lower voltages for better separation of many products and/or long amplicons (>1 kb). Similarly, shorter running times and higher voltages (up to 120V) can be used if you’re in a big hurry, but it’s not ideal and can result in smearing.

Visualize and photograph gel using UV light box, GelDoc, etc
If visualizing with GelDoc, use the following steps:
Use a paper towel to open the drawer of the GelDoc.
Remove the tray from the gel box and carefully slide the gel onto the glass surface of the drawer
Remove one glove, close the drawer and open the main door of the GelDoc. Open the QuantityOne software and select File -> GelDoc XR.
Turn on the white lamp by pressing the Epi White button and click “Live/Focus”
Use your gloved hand to center the gel
Note
You can put a KimWipe along the edge of the gel if it is sliding too much on the glass.


Close the door and turn on the UV lamp (Trans UV button)
Take a picture by clicking either “Auto Expose” or “Manual Acquire”, then “Freeze”. Adjust the exposure length as necessary until you are satisfied with the image.
Note
The brightness of the bands will decrease the longer they are exposed to UV, so keep the lamp on for as little time as possible.

Click “Save,” then “Print.”
Note
The software has a number of really useful features (via “Analyze”) for processing and analyzing the image, which is really helpful if you are doing DNA fingerprinting, multiplex PCR, etc. These can be explored at http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4000126-14A.pdf

Dispose of the gel properly and clean the glass surface of the GelDoc with a paper towel.
Protocol references
Curd, E.E., Gold, Z., Kandlikar, G.S., Gomer, J., Ogden, M., O’Connell, T., Pipes, L., Schweizer, T.M., Rabichow, L., Lin, M., Shi, B., Barber, P.H., Kraft, N., Wayne, R., Meyer, R.S., 2019. Anacapa Toolkit: An environmental DNA toolkit for processing multilocus metabarcode datasets. Methods Ecol. Evol. 10, 1469–1475. https://doi.org/10.1111/2041-210X.13214
Leray, M., Yang, J.Y., Meyer, C.P., Mills, S.C., Agudelo, N., Ranwez, V., Boehm, J.T., Machida, R.J., 2013. A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents. Front. Zool. 10, 34.https://doi.org/10.1186/1742-9994-10-34