Jan 09, 2025

Public workspaceProcessing of red deer samples for Genotyping

DOI
dx.doi.org/10.17504/protocols.io.261gerwwjl47/v1
  • 1Justus-Liebig-University Giessen
Gerald Reiner
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DOI: dx.doi.org/10.17504/protocols.io.261gerwwjl47/v1
Protocol Citation: Julian Laumeier, Corinna Klein, Hermann Willems, Gerald Reiner 2025. Processing of red deer samples for Genotyping. protocols.io https://dx.doi.org/10.17504/protocols.io.261gerwwjl47/v1
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: December 20, 2024
Last Modified: January 09, 2025
Protocol Integer ID: 116606
Keywords: red deer; population genetics
Funders Acknowledgements:
Wildlife and Biotope Conservation Foundation of the North Rhine-Westphalia Hunting Association
Grant ID: 1234
Hessian Ministry for the Environment, Climate Protection, Agriculture and Consumer Protection
Grant ID: 3456
Abstract
The threat of isolation to red deer (Cervus elaphus) has been described in numerous European studies. The consequences range from reduced genetic diversity and increased inbreeding to inbreeding depression. It has been shown that the underlying factors cannot be generalised, but vary greatly in their effects depending on local conditions. The aim of this study was to analyse in detail the genetics of red deer in a large German federal state with a population density of 532 inhabitants per km2 and 23.8% settlement and traffic area, in order to generate data for future management of the region. 1199 individual samples of red deer were collected in all 20 Administrative Management Units (AMUs) and compared with existing results from the neighbouring state of Hesse (19 AMUs). All 2490 individuals from both states were clustered using Bayesian methods and connectivity between neighbouring AMUs was quantified. Overall, 30% of the AMUs were found to be highly isolated, mostly with effective population sizes (Ne) < 100. In contrast, 47.5% of the AMUs still had clear connectivity, allowing them to be merged into 4 larger red deer regions. For the small isolated areas, low genetic diversity was found in unit with high homozygosity and low Ne. With high sampling density and identical methodology, detailed information on AMUs can be obtained and the degree of vulnerability of individual AMUs as part of the overall population can specifically be validated. Such data can help improve future wildlife management.
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Guidelines
All the necessary information can be found in the publication (to be provided as soon as it is published). This is where you will find all the additional details that may help to put this information into practice.
Materials

Equippment

• Steel ball: Retsch GmbH, Haan

• Ball mill: MM 300; Retsch GmbH, Haan

• Ball mill inserts: custom-made for molecular biology; Qiagen GmbH, Hilden

• scalpel: disposable surgical scalpels; BIBRAUN, B. Braun Melsungen AG, Melsungen
• reaction vessels: 2 ml, 1.5 ml; receiver tubes, elution tubes; INSTANT Virus RNA Kit; Analytik Jena AG, Jena
• Reaction vessels: 2 ml, 1.5 ml; nerbe plus GmbH, Winsen/Luhe
• Reaction vessels: 0.5 ml; Brand GmbH und Co. KG, Wertheim
• Reaction vessels: 0.2 ml; Sarstedt AG und Co. KG, Nümbrecht
• Filter vessel: Spin Filter; INSTANT Virus RNA Kit; Analytik Jena AG, Jena
• Vortexer: Vortex-Genie 2; Sceintific Industries, Bohemia, New USA
• Centrifuge: Biofuge fresco; Heraeus Holding GmbH, Hanau
• Petri dishes: 90 mm x 14.2 mm PS without; VWR International GmbH, Darmstadt
• Pipettes: Biohit Deutschland GmbH, Rosbach
• Pipette tips: VWR International GmbH, Darmstadt and nerbe plus GmbH, Wertheim
• Latex gloves: Carl Roth GmbH und Co. KG, Karlsruhe
• PCR plate: AB 1400; Thermo Fisher Scientific GmbH, Dreieich
• PCR plate film: AB 0626; Thermo Fisher Scientific GmbH, Dreieich
• PCR thermal cycler: Tpersonal 48; Biometra, Göttingen
• Magnetic shaker: Variomag Monoshake; Thermo Fisher Scientific GmbH, Dreieich
• Capillary sequencer: ABI Prism 310 Genetic Analyzer; Applied Biosystems, Forster City, California, USA
• Capillary: Polymicro Technologies; Molex; Lisle, Illinois, USA
• Water bath: Type WB 14; Memmert GmbH und Co. KG, Schwabach
• Fluorometer: Qubit‱ Flex Assay Reservoir; Fisher Scientific GmbH, Schwerte

Chemicals and buffers

• Lysis Solution RL; INSTANT Virus RNA Kit; Analytik Jena AG, Jena
• Binding Solution RBS; INSTANT Virus RNA Kit; Analytik Jena AG, Jena
• Washing Solution HS; INSTANT Virus RNA Kit; Analytik Jena AG, Jena
• Washing Solution LS; INSTANT Virus RNA Kit; Analytik Jena AG, Jena
• Proteinase K; Carl Roth GmbH und Co. KG, Karlsruhe
• RNase-free water; INSTANT Virus RNA Kit; Analytik Jena AG, Jena
• ABsolute Blue Q-PCR Rox Mix; Thermo Fisher Scientific GmbH, Dreieich
• Ultrapure water; Rotisolv HPLC Gradient Grade; Carl Roth GmbH und Co. KG, Karlsruhe
• Primer Cervus F/R; biomers.net Gmbh, Ulm
• Primer Cervus S; Probe; biomers.net Gmbh, Ulm
• TE-buffer pH 8.0; TrisHCL 10 mM and EDTA 1 mM; Carl Roth GmbH und Co. KG, Karlsruhe
• 2x Qiagen Multiplex PCR Master Mix; Qiagen GmbH, Hilden
• Orange DNA Size Standard; Mc Lab, South San Francisco, California, USA
• Formamid; Applied Biosystems, Forster City, California, USA
• Gel POP 7 Polymer; Applied Biosystems, Forster City, California, USA
• 3730 Buffer with EDTA; Applied Biosystems, Forster City, California, USA
• Primer; biomers.net GmbH, Ulm
SampleTissue from red deer

Safety warnings
Warnings are given directly in the protocols
Ethics statement
No experimental animals were used for this study. We used only samples from red deering after legal hunting. No animal was sampled or hunted for the purpose of this study.
Before start
Before start make sure that you have a good sample, including
- enough animals per population (we recommend 60)
- enough populations
- a good mixture of individuals from the respective population
Processing of red deer samples
Processing of red deer samples
DNA extraction from tissue samples with Instant Virus RNA Kit delta PREP
Use nitrile gloves!
Preparation of the Virus RNA Kit for first use
(Label kit with expiry date)
  • Mix Washing Solution HS with 70 ml 96-99.8 % ethanol (from the chemical cabinet below the fume cupboard)
  • Mix Washing Solution LS with 144 ml 96-99.8 % ethanol
  • Check off on the container in each case

Preparation for extraction
with 24 samplesà while incubating samples on shaker!
24x Extraction Cups, 72x Receiver Tubes, 24x purple Spin Filters, 24x Elution Tubes for extracted DNA Label all lids with sample numbers

DNA extraction
  1. Select max. 24 samples with tissue (in 2 ml epis) (more will not fit in the centrifuge)
  2. + 500 μl Lysis Buffer RL each (for bone chips 800 - 1000 μl Lysis Buffer RL+ 20 μl Proteinase K)
  3. + one grinding ball each
  4. Load ball mill evenly (CAVE: close both caps, also pay attention to unused side, if < 24 samples insert empty epis in the corners) and run at 25 Hertz/s for 2 min
  5. Remove grinding balls with bar magnets (with glove over) and clean in DNA-Ex
  6. Incubation on the shaker (1000/min) for 30 - 60 min at RT (the longer, the more effective); (for bone chips, incubate for at least 10 h)
  7. Centrifuge for 1 min at 13,000 rpm (for bone chips centrifuge for 3 min)
  8. Pipette off 400 μl of the supernatant and transfer to a new 2ml extraction cup (IMPORTANT: pipette carefully so as not to pick up the pellet, otherwise the Spin Filter membrane will clog later!
  9. + 400 μl Binding Solution RBS (if more supernatant was pipetted off for bone chips, also adjust the RBS volumeà 1:1 (μl supernatant : μl RBS)
  10. Pre-text 10 s (important!)
  11. Pipette 650 μl RL-RBS sample into the prepared Spin Filter plus Receiver Tube
  12. Centrifuge for 1 min at 13,000 rpm (if the solution does not completely pass through the SpinFilter, then centrifuge again, if necessary clean the SpinFilter very carefully with a pipette tip)
  13. Place Spin Filter on a new Receiver Tube, discard used Receiver Tube (liquid in organic waste)
  14. + 500 μl Washing Solution HS
  15. Centrifuge for 1 min at 13,000 rpm (if the solution does not completely pass through the SpinFilter, centrifuge again)
  16. Set Spin Filter to a new Receiver Tube, discard used Receiver Tube
  17. + 500 μl Washing Solution LS Centrifuge for 3 min at 13,000 rpm (if the solution does not completely pass through the SpinFilter, centrifuge again)
  18. Place the Spin Filter in a new 1.5 ml Elution Cup, discard the used Receiver Tube
  19. + 50 μl RNAse-free water (for tissue with a very high DNA concentration such as lung or liver, use 60 μl RNAse-free water for dilution; for muscle samples possibly only 40 μl)
  20. Incubate for 3 min at room temperature
  21. Centrifuge for 1 min at 8,000 rpm
  22. Discard spin filter; DNA is now in 1.5 ml cup

Measurement of the DNA concentration
Qubit Flex
dsDNA Br Kit (Broad Range) or dsDNA Hs Kit (High Sensitivity, with less DNA per sample)
Contains:
  • Buffer
  • Colorant
  • Standard #1 and Standard #2
o Always store in the refrigerator
Ingredients are sensitive to light Always keep in a dark place and store in a cupboard
Total volume in one container always 200μl
Preparation of the DNA measurement:
Standard measurement required? Every +/- 100 samples new measurement with the respective standard (see list at Qubit)à if yes, see below
  1. Light off Always take the kit out of the cupboard for as short a time as possible and work quickly
  2. Gloves on
  3. Prepare number of multistrips
  4. Mix DNA on vortex, if thawed or stood for a longer period of time
  5. Pipette 3 μl per DNA into multistrips
  6. Reagent calculator I. Number of samples II. Standards III. Include surplus: e.g. 2 extra samples
  • For <10 samples, simply calculate one more sample
  • As a reserve for pipetting
  1. Mixing the buffer-dye mixture I. Pour the buffer into a small glass beaker II. Add colorant III. Mix with pipette and swirl
  2. Pipette 197 μl of the buffer-dye mixture to the DNA in each case
  3. Lid closed
  4. Incubate for 2 minutes (if preparation has not already taken a long time)

Testing: dsDNA Br/ dsDNA
  1. Set sample range:
  2. Setting
  • Standard measurement and sample measurement: when a standard is running
  • Without standards: Sample measurement
  1. Deselect unfilled tubes by on them
  2. Select units ng/μl
  3. Measureà CAVE: There must be no air bubbles!
  • Core area: this is where the results should lie
  • Extended range: the values are only targeted here
o If sample is too high then dilute
o If the sample is too small, nothing can be done and it may still be sufficient for capillary electrophoresis (possibly use 2 μl instead of 1 μl DNA for PCR if < 1 ng/μl)
  1. (Data can be found again under Data (Home screen))
  2. Enter the number of samples measured in the table (depending on the buffer) to know when the next standard measurement is required (add to the samples already measured)
  3. Enter results in Excel red deer file
Standard measurement
Every +/- 100 samples: new measurement with the respective standard
Always use the appropriate buffer and dye for the standard! Solutions must be at room temperature
  1. 10 μl each of standard solution #1 in eight tubes (1 multistipe) and 10 μl each of standard solution #2 in eight tubes
  2. Add 190 μl buffer dye mixture to each
  3. Measure: Main menu again dsDNA Br/ dsDNA Hs -> Read standards and measure samples
  4. Enter standard measurement in list
DNA dilution
  1. Copy the corresponding sample numbers in the Red Deer Excel table and paste the values on the "PCR assays" page twice for each PCR number (once for MM1 and once for MM2)
  2. Leave the first field blank for the PCR standards (BKW_059)
  3. Enter measured DNA content
  4. Automatic calculation of the required dilution (target: 2.5 ng/μl)à CAVE: possibly set to a target of 5 ng/μl
  5. Label 0.5ml cup
  6. Mix DNA in vortex
  7. Add the calculated amount of RNAse-free water (from DNA extraction kit) to Eppis; if there is a number in the column next to H2O (>5:250) (if there is > 250 μl H2O to be added), then first dilute 5:45 (= 1/10) and then the number next to it (5:x); this saves RNAse-free water.
  8. Pipette 5 μl of the respective DNA into the tubes (V(DNA) = 5 μlà if less, pipetting error is too high).
PCR approach
Check whether there is enough BKW59 standard (in the freezer of the capillary sequencer room) or whether it also has to go into the PCR
First of all: Place the master mixes in the refrigerator in the "Sample addition" room to defrost!
A maximum of 24 samples can be processed per PCR device (23 samples are possible in the capillary sequencer)
Place the rack with the DNA in the "PCR preparation" room in the refrigerator.
Space "Sample addition"à No DNA here!
In the freezer, usually on the top shelf, there is a box labeled "new MS Hirsch (for Kap. Sequencer)"
  1. Check the volume of the mastermixes or primer mixes (mastermixes: MM H1 and MM H2). Is there enough primer left?à Draw up the required amount with a pipette and check whether enough is drawn up
a. If not, then prepare new mastermixes from the individual forward and reverse primers: Instructions in Oligo folder under "Capillary sequencing deer" (mix primer mixes by snipping, then piepette primer mixes with water and mix 2xMM
b. If you have Eppendorf tubes labeled MM H1 and MM H2, take them out and put them in the refrigerator to thaw, so do this first!
  1. Label 0.2 ml single tubes in PCR tray without lid 2x 23 (23 per master mix; e.g. first 23 sample numbers in black, second 23 sample numbers in green
  2. Pivot MasterMix
  3. 9μl MM1 in the first half (1-23) of the
  4. 9μl MM2 into the other half (24-46) of the tubes
  5. Put the master mixes back in the freezer
  6. Change room to "PCR preparation" room (take PCR trays with 46 tubes)
Now switch on the PCR device for preheating, as it takes approx. 20 minà see below
  1. Mix the DNA sample (flick it and then shake it down)
  2. 1μl DNA in each tube (if < 1μl DNA is measured, you can also use 2 μl) with shrink-wrapped filter pipette tips; 2x each, as two master mixes
  3. Mix the DNA with the master mix: Place lid on to mix and invert until liquid collects in the lid, then knock back down into the tip
Example for PCR run with 23 samples
Sample 1 Pr. 2 Pr.3 Pr.4 Pr.5 Pr.6 Pr.7 Pr.8
MM H1 MM H1 MM H1 MM H1 MM H1 MM H1 MM H1 MM H1
Pr.9 Pr.10 Pr.11 Pr.12 Pr.13 Pr.14 Pr.15 Pr.16
MM H1 MM H1 MM H1 MM H1 MM H1 MM H1 MM H1 MM H1
Pr.17 Pr.18 Pr.19 Pr.20 Pr.21 Pr.22 Pr.23 Possibly Pr.24
MM H1 MM H1 MM H1 MM H1 MM H1 MM H1 MM H1
Pr. 1 Pr. 2 Pr.3 Pr.4 Pr.5 Pr.6 Pr.7 Pr.8
MM H2 MM H2 MM H2 MM H2 MM H2 MM H2 MM H2 MM H2
Pr.9 Pr.10 Pr.11 Pr.12 Pr.13 Pr.14 Pr.15 Pr.16
MM H2 MM H2 MM H2 MM H2 MM H2 MM H2 MM H2 MM H2
Pr.17 Pr.18 Pr.19 Pr.20 Pr.21 Pr.22 Pr.23 Possibly Pr.24
MM H2 MM H2 MM H2 MM H2 MM H2 MM H2 MM H2
Room change
  1. Switch on the PCR device, if not already done (always takes a while to heat up): Start/Stopà Select the program from the list and enter the number (Hermannà Program 20 (HIRSCH 60))à Enter (program is now running);
Only preheat and do not start PCR yet: additionally start/stop afterwardsà Pause
  1. Adjust samples (if full, remove empty tubes, otherwise leave in the corners), close and screw shut
  2. If preheated: Start/Stopà "Continue"; then the program runs (approx. 2h)
  3. Get new PCR sample racks with lids and label them for later freezing
  4. Check whether the correct program is running, "Info" shows the duration it still
When PCR ready:
  1. Flashes "Pause" and 4°
  2. Open the lid and take out the samples
  3. Switch off the device
Denaturation
  1. Remove the formamide with O standard from the freezer and allow to thaw in the dark (protect from light); (if no formamide with O standard is ready mixed, then mix the prepared 1200 μl formamide with 15 μl O standard)
  2. Preheat the water bath to 95°C
  3. Sort 48 x 0.5 μl Eppi into rack (CAVE: where is 1?) and label; standard is Eppi 1 and 25
  4. Place 12μl formamide in each Eppi
  5. Add 1 μl BKW059 standard each to Eppi 1 and Eppi 25
  6. Add 1μl of the respective PCR DNA samples to the remaining Eppis and then place on a new small blue rack, always leaving a hole free (6 tubes per row)
  7. Place the rack with tweezers in the water bath at 95° for 3 minutes so that the rack floats
  8. Briefly drain the rack and immediately place in the freezer for 2 minutes
  9. Switch off the water bath
  10. Formamide back in the freezer
  11. Sequencing or freezing samples (small blue rack in purple box)
Capillary sequencing
CAVE: first test/rinse run if capillary sequencer has not been run for several days (run any sample)
  1. Start up PC
  2. Open the sequencer and remove the purple box!
  3. Switching on the sequencer
  4. Start ABI Prism310 program on PC
  5. (Option to save 20 minutes: Preheat temperature to 60°C: Windowsà Manuel controlà Temperature setà 60à Execute Create sample sheet
a. New Genscan Sample sheet 48
b. Set 5 dyes at the top right (default with "diamond" must be orange)
c. Enter sample name
d. Close Save As Naming the injection list
  1. Set samples
a. Open sequencer
b. Press "Tray"
c. Sorting samples CAVE: A1 rear right
d. Retract tray with the same button
  1. IMPORTANT: Check whether there is enough gel in the glass syringe (> 0.2 ml), if too little, see below
  2. IMPORTANT: Fill buffer in container 1 up to the black line; fill Roth water in container 2 up to the black line; fill Roth water up to the edge of Eppi in position 3
  3. Start run
a. New Genscan Injection List
b. sample sheet just created
c. Insert "Laser off" last in the open file for KS2!!! (not necessary for KS1)
Moduleà Laser off.md5
i. If 48 samples then: Edità Add (inserts new line)
ii. Set moduleà Laser off md.5
iii. And give the sample a name (any name)
d. RUN (takes approx. 19 hours)
If gel < 0.2, then it must be topped up:
  1. Pop7 Aliqu. Remove the buffer from the top freezer compartment and defrost at approx. 50° in an ultrasonic bath
  2. In program ABI Prism310: Window Manual Controlà Syringe Home (moves up)
  3. Shift syringe drive toggle to the left
  4. Unscrew the gel syringe and fill it bubble-free with Pop7 Aliqu. Buffer up to 0.7-0.8ml
  5. Screw the gel syringe back in; turn the waste valve carefully and, if necessary, press the glass syringe down slightly until all the air bubbles run to the left or down over the "crossing"; then tighten again
  6. Move syringe drive toggle to the right
  7. On the computer: Syringe down in careful steps until the syringe drive is lightly applied
Sampling and data collection
  1. When Run is finished, close the Run window/injection list and save it
  2. Close program
  3. Drag Run in Explorer onto the USB stick
  4. Shut down PC
  5. Move tray out and remove samples; provide samples with labeled lids and freeze in bags
  6. Move tray
  7. Push the purple box under the autosampler!
  8. Switch off the device