Annex

10X PCR Buffer (conventional)

100mM Tris-HCl (pH 8.8 at 25°C), 500mM KCl, 0.8% Nonidet P40.

10X PCR Buffer with (NH₄)₂SO₄

750mM Tris-HCl (pH 8.8 at 25°C), 200mM (NH4)2SO4, 0.1% Tween 20.

Extraction solution

Extraction solution was 0.1 M EDTA, 0.2 M NaCl, 0.05 M Tris-HCl (pH 8.0), 0.5% SDS

loading dye solution, 6X

1 0.09% bromophenol blue, 0.09% xylene cyanol FF, 60% glycerol, 60mM EDTA and appropriate volume of water. Store at –20ºC.

2 0.25% bromophenol blue, 40% w/v sucrose in water. Store at 4ºC.

PCR water

Autoclave double distilled water and expose it frequently to UV in a lamina flow cabinet.

Phenol, buffered

Add 0.5 g of 8-hydroxyquinoline to 500 ml melted phenol and add 500 ml of 50mM Tris base. Stir 10 min at low speed, let phases separate and decant the aqueous. Add 500 ml of 50mM Tris-Cl pH 8.0 and keep on washing until pH 8 of decanted solution is achieved. Add 250 ml of TE buffer and store <2 months at 4ºC.

Phosphate buffered saline

For 1 liter 10X stock; 80 g NaCl, 2 g KCl, 11.5 g Na₂HPO₄.7H₂O and 2 g KH₂PO₄ dissolved in distilled water up to 1 liter.

Primer solution

Primers were supplied in solid form, 50mM solutions of all six primers were made by dissolving the primer in appropriate volume of PCR water.

Primer IBB first left solution

77 nano mol primer of molecular weight 5357 (4.1249 x 10^-4g) plus 1540 µl water.

Primer IBB first right solution

66 nano mol primer of molecular weight 5437 (3.58842 x 10^-4g) plus 1320µl water.

Primer IBB second left solution

77 nano mol primer of molecular weight 5433 (4.1834 x 10^-4g) plus 1540 µl water.

Primer IBB second right solution

64 nano mol primer of molecular weight 5130 (3.2832 x 10^-4g) plus 1280 µl water.

Primer IBB third left

82 nano mol primer of molecular weight 5198 (4.2624 x 10^-4g) plus 1640 µl water.

Primer IBB third right

78 nano mol primer of molecular weight 5742 (4.4788 x 10^-4 g) plus 1560 µl water.

TBE buffer

Composition of 10X stock solution / 500ml: 54 g of Tris base, 27.5 g of boric acid, 20 ml of 0.5 M disodium salt of EDTA (pH 8.0).

TE buffer

10 mM Tris-Cl, pH 7.4, 7.5 or 8.0 and 1 mM EDTA, pH 8.0