TITLE: ABI Sanger Sequencing of Avian Clock genes to elucidate markers for Migration Phenology
AUTHORS: Louis-Stéphane Le Clercq, Desiré Lee Dalton, Antoinette Kotzé, Paul Grobler

[DESCRIPTION]
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.

[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.

[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 2880 min  until analysis.
Briefly vortex reagents and mixes as needed.


[STEPS]
SECTION: Big Dye Master Mix setup
1. Prepare   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)
SECTION: Cycle sequencing
2. Program and run the following cycle conditions on a thermal cycler, e.g. 
 
Initial denaturation at 96 °C  for 1 min 
25 cycles of:
Denaturation at 96 °C  for 10 s 
Annealing at 50 °C  for 5 s 
Extension at 60 °C  for 2 min  
Hold at 4 °C  until next step.

SECTION: Sequence reaction clean-up
3. Purify the sequencing products using   

SECTION: Capillary electrophoresis &amp; Data capture
4. Load sequencing reactions to sequencing plate and set up a run on the genetic analyzer, e.g. 

  
 
  
Export sequence read trace files once done!
Files can be opened with 
 
or
 
or

 

 



SECTION: Big Dye Master Mix setup
1.1. Prepare the following sequencing master mixes in duplicate, one for each primer (forward and reverse).

Master Mix:

 

  

SECTION: Big Dye Master Mix setup
1.2. Add 9 µL  to 1 µL  to the individual wells of a 96-well PCR plate or thin-walled PCR tubes.

SECTION: Sequence reaction clean-up
3.1. Vortex the bottle of BigDye XTerminator™ beads for 8 to 10 seconds before mixing with the SAM solution.
SECTION: Sequence reaction clean-up
3.2. Prepare the SAM/BigDye XTerminator™ bead working solution:

  

SECTION: Sequence reaction clean-up
3.3. Transfer the indicated volume of bead mix (BigDye XTerminator™ bead solution and SAM solution) to each.
SECTION: Sequence reaction clean-up
3.4. Vortex the 96-well plate/tubes  at    on a shaker, e.g. 
 


SECTION: Sequence reaction clean-up
3.5. In a swinging bucket centrifuge, centrifuge the plate at 1000 x g, 2 min, 21 °C   .