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You are here: Home Resources Behavior WHO IS WHO? NON-INVASIVE METHODS TO INDIVIDUALLY MARK AND SEX CHICKS

WHO IS WHO? NON-INVASIVE METHODS TO INDIVIDUALLY MARK AND SEX CHICKS

  Iris Adam*, Mariam Honarmand*, Constance Scharff

          Department of Animal Behaviour, Freie Universtität Berlin

 

 

1. Marking of newly hatched chicks

 

Down-feathers of newly hatched chicks are trimmed using a fine pair of scissors. Markings are applied following hatching order:

A= head

B= back

C= wings

D= sides

E= none

If there are more than 5 chicks hatch in one nest, apply combinations e.g.

F= head + back

G= sides + wings

Make sure to trim down all the downs of a patch, otherwise it is difficult to recognize the markings at later time points.

 

2.  Scharff lab PCR sexing protocol 

 

Sampling

  1. Hold a piece of filter paper using a forceps and take a swab from the inside of the beak of the bird. Try to contact the mucous membranes in the beak, tounge and choana to collect epithelial cells. Place the piece of filter paper into a reaction tube containing 200µL of 5% (w/w) Chelex100 (BioRad 142-1253).
  2. Blood: Take a small canula and puncture either the wing vein or the leg vein. Sample a tiny droplet of blood with a piece of filter paper held with a forceps. Drop the piece of filter paper into a reaction tube containing 200µL 5% (w/w) Chelex100.

 

DNA-extraction:

  1. Incubate the tube at 56°C for 15min in a heat block.
  2. Vortex for approx. 10s.
  3. Boil for 8min in heat block.
  4. Vortex for approx. 10s.
  5. Centrifuge at 15,000 g for 3min.

 

PCR:

  1. Prepare one 0,2mL tube for each bird plus one for a positive and one for a negative control.
  2.  Prepare a master mix according to the recipe below. Make 10% more than needed to allow for pipetting losses. Keep on ice.
  • For DNA from blood add 10.8µL water per sample.
  • For DNA from saliva, do not add any water to the reaction.

3. Distribute the master mix into the prepared PCR-tubes.

 

25µL sample

1x

x

 

TDM

4µL

 

 

Primer P2 (100 pmol/µL)

0.8µL

 

 

Primer P8 (100 pmol/µL)

0.8µL

 

 

Taq

0.3µL

 

 

H2O

11.1µL/ 0µL

 

 

DNA

8µL/ 19.1µL

 

 

 

  1. To each tube with master mix, add 8µL (blood) or 19.1µL of the DNA-solution. Add water to the negative control (No Template Control=NTC).
  2. Keep the samples on ice until you place the tubes in the PCR-cycler and run the sexing program (see below).

 

94°C

5min

 

94°C

30s

 

55°C

30s

 

72°C

45s

Repeat 40 times

72°C

5min

 

4°C

Forever

 

 

Run PCR-product on a 2% agarose gel.

 

Material:

 

Filter paper:

E.g. Whatman Cellulose Chromatography Papers Grade 3MM Chr (3030-917)

Normal office paper does not work.

 

Chelex:

Bio-Rad (100-200 mesh) Cat-Nr: 142-2832 (You can ask for a free test sample.)

 

Primer:

We are using the P8 P2 combination from Giffiths et al. 1998 [1].

 

Taq:

We are using homemade Taq (10u/µL).

 

TDM:

 

Stock concentration

For 1mL

10x Pcr-buffer without Mg2+

10x

677.56µL

dNTP

25mM each

48.11µL

MgCl2

50mM

274.31µL

 

1.       Griffiths, R., M.C. Double, K. Orr, and R.J. Dawson, A DNA test to sex most birds. Mol Ecol, 1998. 7(8): p. 1071-5. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9711866