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.