TITLE: PCR Amplification of Clock and Adcyap1 genes with EmeraldAmp® GT PCR Master Mix in Avian species for polymorphism elucidation
AUTHORS: Louis-Stéphane Le Clercq, Desiré Lee Dalton, Antoinette Kotzé, Paul Grobler

[DESCRIPTION]
This PCR protocol is used to amplify Clock and Adcyap1 gene regions in avian species which have previously shown polymorphisms, such as poly-Q runs, that correlated to migration phenology. It was tested and optimized in Woodlands kingfisher (Halcyon senegalensis) and Diederik cuckoos (Chrysococcyx caprius). The primers were designed based on those previously used by Johnson et al. (2007) and Steinmeyer et al. (2009) by comparing the relevant gene sequences for chickens (Galus galus) with several other available avian species to select primers that would account for the most common variations in primer regions, enabling more universal amplification. Individual clock gene sequences were retrieved from Genbank and aligned in BioEdit 7.2. Primers were then selected based on the annotated regions. The assay was designed using 25 μL (half) reactions of EmeraldAmp® GT PCR Master Mix, which is premixed with loading buffer for easy gel loading following PCR and does not require a long initial denaturation step (thereby shortening the run time). Gel electrophoresis was able to confirm successful amplification of a product ±280 bp long in both species. The same primers were subsequently used for sanger sequencing. A BLAST search of the resulting sequences confirmed the identity of the amplified regions.

[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 PCR worksheet template is included for download to automatically calculate volumes.
Check DNA template quantity and purity prior to use in PCR.
  allows for direct gel loading.
Equipment used are interchangeable with industry equivalents. 
Experiments performed at Room temperature  is always at 21 °C .
Treated PCR products may be stored at -20 °C until required for sequencing.
Briefly vortex reagents and mixes as needed.



[STEPS]
SECTION: Master Mix set-up
1. Prepare Master Mix and Samples* for PCR.

*Sample information has been deposited to BioSample and associated to the BioProject (PRJNA737185) which used this protocol.

(An experiment template is included in excel format.)
SECTION: Thermal cycling
2. Program and run the following thermal cycling profile on a thermal cycler, e.g. 
 
40 Cycles  of:

Denaturation at 98 °C  for 10 s 
Annealing at 60 °C  for 30 s 
Elongation at 72 °C  for 1 min 

Infinite hold at 4 °C  until ready for next steps.

  

SECTION: Electrophoresis
3. Confirm success of amplification by TAE/TBE electrophoresis.
SECTION: Amplicon purification
4. Purify the positive amplicons with   prior to sequencing.

SECTION: Master Mix set-up
1.1. Set up the following Master Mix with    for a 25 µL reaction in a DNA-free lab and laminar flow cabinet.
 
    

SECTION: Master Mix set-up
1.2. Add 23 µL to 2 µL* in individual thin-walled PCR tubes in a DNA-loading laminar flow cabinet.

  

*DNA isolated from avian blood samples may be highly concentrated, ensure that it is still less that 500 ng  of DNA per reaction.

SECTION: Electrophoresis
3.1. Prepare a 2 % (v/v)  gel with   and   , pre-stained with   using a casting tray and comb with sufficient wells.

 Resolution capacity of different concentrations of gels.
                    Concentration   of Agarose  Efficient range of   separation of linear DNA (kbp)    0.3  5 to 60 kbp    0.6  1 to 20 kbp    0.7  0.8 to 10 kbp    0.9  0.5 to 7 kbp    1.2  0.4 to 6 kbp    1.5  0.2 to 3 kbp    2.0  0.1 to 2 kbp  
 
 Amount of agarose required for a small (50 mL) and large (100 mL) gel.
                0.3  150mg  300mg    0.6  300mg  600mg    0.7  350mg  700mg    0.9  450mg  900mg    1.2  600mg  1.2g    1.5  750mg  1.5g    2.0  1g  2g  
 
*Note:  is usually added at a concentration of 1 µL  per 10 mL 
  

SECTION: Electrophoresis
3.2. Load 4 µLof PCR product to the gel alongside a molecular weight marker, e.g.  , and run at 60 Volt for 30 min . Possible settings for the 
 
are:

 


SECTION: Electrophoresis
3.3. Visualize and capture gel on an appropriate imager and paired software, e.g. 
 

 

SECTION: Amplicon purification
4.1. Mix 5 µL  with 2 µL  for a total volume of 7 µL .

SECTION: Amplicon purification
4.2. Incubate at 37 °C  for 15 min  to degrade PCR primers and short products. 

SECTION: Amplicon purification
4.3. Incubate at 80 °C  for 15 min  to inactivate the   .
  

SECTION: Amplicon purification
4.4. The PCR product is now ready for use in DNA sequencing*, SNP analyses, or other primer-extension applications.

*See Clock genes sequencing protocol (https://protocols.io/view/abi-sanger-sequencing-of-avian-clock-genes-to-eluc-bvydn7s6)