APPENDiX I

Agarose gel

Agarose (SRL) 1.0g

IX TBE (see TBE butTer) lOOm!

The agarose was melted in the microwave oven and allowed to cool to 50°C

before pouring the gel.

Bromophenol blue loading dye

Bromophenol blue 0.05 g

Glycerol 5.0 ml

EDTA 0.186 g

1 X TAE volume to 10 ml

The loading dye was dispensed in 1 ml aliquots in fresh Eppendorf tubes and

stored at 4(lC.

Disodium ethylenediamine tetraactete (EDT A) - 0.5 M

EDTA 186.!g

Distilled water

NaOH

1000 ml

-20 g

EDT A was dissolved in 800 ml of distilled water and stir vigorously 011 a

magnetic stirrer. Adjust pH to 8.0 using NaOH pellets and make the final volume to

1000 m!. Autoclave before use.

Ethidium Bromide (10 mg/ml)

Ethidium Bromide 0.1 g

Distilled water 10ml

The solution was stirred using a magnetic stirrer for several hours to ensure

that the dye had completely dissolved. The container was wrapped in aluminium foil

and stored at 4()C For staining agarose gels, a working solution of 0.5 Ilgiml was

made.

Hexadecyltrimethylammonium bromide (CT AB) - 10 % in 0.7 M l\aCl

CTAB 109

NaCl

Distilled water

4.1 g

80 ml

Dissolve 4.1 g NaCI in 80 ml distilled water and slowly add 10 g eTAB,

while heating and stirring. If necessary, heat to 65"C to dissolve. Adjust final volume

to 100ml.

Oxidase reagent

Tetramethyl p-phenyldiaminc

lsoamyl alcohol

Proteinase K (20 mg/ml)

Ig

100ml

Proteinase K 10 mg

Sterile distilled water 0.5 ml

Stored at -20°C in 50 III aliquots.

Sodium dodecyl sulphate (SDS) - 10%

SDS 10 g

Distilled water IOOml

Tris-Acetate EDT A (50 X)

Tris base

0.5 M EDT A (pH 8.0)

Glacial acetic acid

Distilled water

242 g

100ml

57.1 ml

to 1000 ml

The stock solution was diluted to I X for gel runs.

Tris-Borate EDT A (TBE) buffer - 5 X

Tris base

Boric acid

0.5 M EDT A (pH 8.0)

Distilled water

54 g

27.5 g

20ml

to 1000 ml

The stock solution was diluted to 0.5X just before use as gel running buffer.

Tris EDT A (TE) buffer

Tris-HCI

EDTA (pH 8.0)

Distilled water

10 mM

!mM

to 200 ml

2 ml ! M Tris-Hel (pH 8.0)

0.4 ml 0.5 M EDT A

APPENDIX 11

GenBank submissions

DQ241795 - 16S rRNA Gene from strain FK4, Vibrio alginolyticus.

DQ898173 -16S rRNA Gene from strain EKM14, Vibrio cholerae.

DQ904445 - 16S rRNA Gene from strain PV8(2), Vibrio sp.

DQ904444 - 16S rRNA Gene from strain CHV4(2), Vibrio alginolyticus

strain RH2.

DQ904443 - 16SrRNA Gene from strain MUS13, Vibrio furnissii (AT CC

35016T).

DQ904442 -16S rRNA Gene from stain C4, Vibrio sp.

DQ904441- 16S rRNA Gene from strain PlO, Vibrio .\p.