Nov 23, 2023
ABI Sanger Sequencing of Avian Clock genes to elucidate markers for Migration Phenology
- Louis-Stéphane Le Clercq1,2,
- Desiré Lee Dalton3,
- Antoinette Kotzé1,2,
- Paul Grobler1
- 1University of the Free State;
- 2South African National Biodiversity Institute;
- 3Teesside University
Protocol Citation: Louis-Stéphane Le Clercq, Desiré Lee Dalton, Antoinette Kotzé, Paul Grobler 2023. ABI Sanger Sequencing of Avian Clock genes to elucidate markers for Migration Phenology. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4k6zrvo5/v1
Manuscript citation:
Le Clercq, L.S., 2023. Biological clock measures: Assessing the association between the circadian and epigenetic clock as predictors of migration phenology and biological aging in wildlife (Doctoral thesis, University of the Free State).
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: June 20, 2021
Last Modified: November 23, 2023
Protocol Integer ID: 50917
Keywords: BigDye, Sanger, DNA sequencing, clock, adcyap1, avian
Funders Acknowledgement:
National Research Foundation (RSA)
Grant ID: 112062
Abstract
This protocol follows up on "PCR Amplification of Clock genes with EmeraldAmp® GT PCR Master Mix in Avian species" and is intended to provide the next steps used in the sanger sequencing of the produced amplicons. This protocol uses ABI BigDye reagents (but suitable alternatives exist). The same primers used to produce the PCR products, of the respective clock genes, are used individually in a forward and reverse sequencing reaction. Cycling conditions mimic those used for PCR. Sequencing reactions are purified and subsequently resolved on an ABI Genetic Analyzer. The sequence read data was used in a BLAST search and confirmed to be the genes and regions of interest for all tested species.
Attachments
Guidelines
- A sequencing worksheet template is included for download to automatically calculate volumes.
- Equipment used are interchangeable with industry equivalents.
- Experiments performed at Room temperature is always at 21 °C .
- Plates can be stored for up to 48:00:00 until analysis.
- Briefly vortex reagents and mixes as needed.
Materials
Reagents:
- BigDye™ Terminator v3.1 Cycle Sequencing KitApplied Biosystems
- Primers: (Inqaba Biotech. Industries)
| A | B | C | D | E | F | |
| Adcyap F | GATGTGAGTAACCAGCCACT | Adcyap1 | Gene ID: 408251 | 20 | 61.3 | |
| Adcyap R | ATAACACAGGAGCGGTGA | Adcyap1 | Gene ID: 408251 | 18 | 59.7 | |
| Clock F1 | TGGAGCAGTAATGGTACCAAGTA | clock | Gene ID: 373991 | 23 | 62.9 | |
| Clock F2 | TGGAGCGGTAATGGTACCAAGTA | clock | Gene ID: 373991 | 23 | 65.0 | |
| Clock R1 | TCAGCTGCGACTGAGCTGG | clock | Gene ID: 373991 | 19 | 66.0 | |
| Clock R2 | TCAGCTGTGGCTGAGCTGG | clock | Gene ID: 373991 | 19 | 66.1 |
Summary of primer details for the assay including the primer name, sequence, gene, gene ID, length and Tm
- BigDye XTerminator™ Purification KitThermo FisherCatalog #4376484
Equipment:
Equipment
SimpliAmp Thermal Cycler
NAME
PCR
TYPE
Applied Biosystems
BRAND
A24811
SKU
LINK
Any standard PCR thermocycler will suffice
SPECIFICATIONS
Equipment
IKA MS 3 Digital Vortex Mixer
NAME
Vortex mixer
TYPE
IKA
BRAND
3319000
SKU
LINK
Vortex mixing of plates
SPECIFICATIONS
Equipment
3500 Genetic Analyzer
NAME
Sequence analyzer
TYPE
Applied Biosystems
BRAND
4440470
SKU
LINK
DNA sequence fragment analysis
SPECIFICATIONS
DNA Amplicons:
- BioSample information information has been deposited to the BioProject (PRJNA737185) linked to this protocol.
Protocol materials
BigDye™ Terminator v3.1 Cycle Sequencing KitApplied Biosystems (ThermoFisher Scientific)
Materials, Step 1
BigDye XTerminator™ Purification KitThermo FisherCatalog #4376484
Materials, Step 3
Safety warnings
- Set up master mixes in a "DNA-free" room and laminar flow cabinet.
- Add DNA to reaction tubes in a "DNA-loading" laminar flow cabinet.
- Always dispose of biohazardous waste appropriately in accordance to lab regulations.
- Always wear gloves and a lab coat.
- Never directly look at the UV lamps.
Ethics statement
Protocol approval for the present study was obtained from the protocol committee of the Department of Genetics, University of the Free State (approval number: Res18/2020). Ethics approvals were obtained from the University of the Free State (approval number: UFS-AED2020/0015/1709) as well as the South African National Biodiversity Institute (approval number: SANBI/RES/P2020/30). Appropriate research permits were also obtained from South African regulatory authorities including the Department of Agriculture, Land Reform, and Rural Development (Section 20 permit: 12/11/1/1/18(1824JD)).
Before start
- Thaw reagents On ice .
- Wipe workspace with 10 % volume Bleach, followed by 70 % volume Ethanol, and ddH2O before (and after).
- UV the relevant laminar flow cabinets.
Big Dye Master Mix setup
Big Dye Master Mix setup
Prepare BigDye™ Terminator v3.1 Cycle Sequencing KitApplied Biosystems Master mix and Samples* for Sanger Sequencing.
*Sample information has been deposited to BioSample and associated to the BioProject (PRJNA737185) which used this protocol.
(An experiment template is included as an excel spreadsheet)
Prepare the following sequencing master mixes in duplicate, one for each primer (forward and reverse).
Master Mix:
| A | B | C | D | |
| BigDye™ 3.1 Ready MM | X2.5 | X1 | 4 | |
| Primer | 3.2 µM | 3.2 pM | 1 | |
| Nuclease free water | - | - | 4 |
Components of sequencing reaction, indicating stock and final concentrations as well as the relative volume needed in microliters. (for a 20μL reaction you can double the volume of each component)
Plate set up for Sanger sequencing.
Add 9 µL Master Mix to 1 µL purified PCR product to the individual wells of a 96-well PCR plate or thin-walled PCR tubes.
Cycle sequencing
Cycle sequencing
1m
Program and run the following cycle conditions on a thermal cycler, e.g.
Equipment
SimpliAmp Thermal Cycler
NAME
PCR
TYPE
Applied Biosystems
BRAND
A24811
SKU
LINK
Any standard PCR thermocycler will suffice
SPECIFICATIONS
- Initial denaturation at 96 °C for 00:01:00
- 25 cycles of:
- Denaturation at 96 °C for 00:00:10
- Annealing at 50 °C for 00:00:05
- Extension at 60 °C for 00:02:00
- Hold at 4 °C until next step.
3m 15s
Sequence reaction clean-up
Sequence reaction clean-up
Purify the sequencing products using BigDye XTerminator™ Purification KitThermo FisherCatalog #4376484
Vortex the bottle of BigDye XTerminator™ beads for 8 to 10 seconds before mixing with the SAM solution.
Prepare the SAM/BigDye XTerminator™ bead working solution:
Volumes of SAM solution and beads to add
Transfer the indicated volume of bead mix (BigDye XTerminator™ bead solution and SAM solution) to each.
Vortex the 96-well plate/tubes at 1800 rpm, 21°C, 00:20:00 on a shaker, e.g.
Equipment
IKA MS 3 Digital Vortex Mixer
NAME
Vortex mixer
TYPE
IKA
BRAND
3319000
SKU
LINK
Vortex mixing of plates
SPECIFICATIONS
In a swinging bucket centrifuge, centrifuge the plate at 1000 x g, 21°C, 00:02:00 .
2m
Capillary electrophoresis & Data capture
Capillary electrophoresis & Data capture
Load sequencing reactions to sequencing plate and set up a run on the genetic analyzer, e.g.
Genetic analyser with plates loaded for capillary electrophoresis.
Equipment
3500 Genetic Analyzer
NAME
Sequence analyzer
TYPE
Applied Biosystems
BRAND
4440470
SKU
LINK
DNA sequence fragment analysis
SPECIFICATIONS
Set up plate layout for fragment analysis on genetic analyser.
- Export sequence read trace files once done!
- Files can be opened with
or
Software
MEGA
NAME
or
Expected result
View of trace file in Seq Scanner 2