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:*

APPENDICES

App.ndtx A
Gaouft LCa
1. ll&L A.dl
Nuflont brD0t (OoH)
Dtddvr.br
agl'.d d .l2t oC tbr 20 t|t||.
2. llutld t{rr
Mlttntdt(OoU)
A€ir agef (Olotd)
DL{lstft
sbdtr.d at 12t qc ft. m |r5.
,t9.0 g
tom il
t3.09
15.0 g
md
3. Uft a.tlla Btrlb
8.6T4fioo. (Oxoit)
Btcio-Y..rt€,faot(o@ld) 59
N€
thfld||t|'
t0 s
'os
io(I}m
pH wa! .dlu3bd io 7.0 ulh l0 M t&OH t tb|b €i.dtdng .[ t2t .C h
2() mhuaa Foa parparalioo of t-|ld*AdEj af|l ,nrrtrrlh GA agra
r:dlm), t50e.rT.r(o,o5)rE d.t ,t h.tur.bbn

2:t3
a. aHt
B|ll 379
160 n{
SbrilE€d Et 121 oC fer.2o ntn, For pEparatbn of BHt e!€r medluG t5 g
ag!' .gir (ol!o5) E dd.d !66.! rtdEddi.
t YE r.dh|||
Bo.b.Ya6toffi
x&Po.
trtgso4. 7!to
(Nrul9O.
5g
Dhtiltad tN€tar
20g
5g
3g
2g
lmoml
pH rra! adFli.tt b 5.0 b*r€ aistadoo at t2l cC for 20 nh. SrEu
frr .6.IiEd .epanbty .t fio t ft. tS fih. Fo. &Id nEdh4 t5 g
agEr eger wa! edd€d b.fio|! rt$tEeton.
o
BAH fl.dtum
B.cto.ycart oxlect
N..tlPO.
Cnrlc !dd. H2O
Dldld ht6.
20g
5g
5g
2.7I
1.15 g
to(tr d

254
pH was adiusi8d io 5.0 b.tore st€dttsaton ar i2t 6C. Gtuco.6 ra3
sleiliz€d €spa€tety at ljo oC for t5 min. For solid mediJm. 15 g agar
agar w.s add6d b€for€ stedtiza uon.
Ttu{luconat. M€dtum
Tds (hydoxrn€thr) aminorn6hane
NaCl
KCI
NTLq
6.06 g
4.64S
1.49 g
1.O7 9
0.439
Mgclr. 6HrO
CaCh_ 2H1o
0.209
0.039
0.04 g
Asar agar (Oxoid)
15.0 g
2.o s
l0O0 ml
pH wac adju3t€d to 7.0 with 2 M HCtand srgritiz€d at j21 oC for 20 min.
Na. Gluconat€ viBs sradtized s€parat€ty al i i O oC for 15 min.
a. KN ||odlum tor EPs pro.tE{on
20g
Eado-Yeast od|dct
KH,PO.
o5s
6.8 9

l(:llFOr
Nact
MgaOl?HrO
DirLd 6.
&8C
o.t c
O.2g
t0(tr d
Elh€r glucot€ o. tiucb|€ of €{rdD!! lr€3 us€d aa cerbon &uto.
Ctton ntco mr .b.t d ..pa*ty rt l tO cC ft. lO r|| e|d ura
w., abo aaparably *.tb.d !t flt ton t.hg 022 lrn nttpo|! ftr.
*l ms e{ded b 70 0€tbr tbdtfig d t2l t ftr 20 d.. For &ad
nbdiint 'l5 C qar {fi 6 €r$sd bafta€ sffior

256
Appendix B
R€agents Used In Quallt ve and euen tativ. anall|.l3 of
Blopolymers
L Phenol-Sutturlc Actd tU€tho.l
. obtiled phonoi, 5 % (w/v) in disriled w5l€i
. Concontrat6d H2SO1,
. Sr.ndard Stucose sotutbn (100 pSr'mD.
2. Cffb.zol. M.ihod
. Concedrabd HrSO. (98 ml) \NBs add€d ro sodium telraborals
docahyd€t6d (0.95 g tn 2.0 mt distil€d watar) and sto€d at 40
'c.
. Calbazole (bcfysb insd fmm absoture €thanot; j2S rp
dassoh€d in 100 mtabsotuia erhanot).
3. Br.dtord ilothod
cooma.ste Ro.gont (1 00 mgt)
100 mg Coomassie Bdttiant Btu6 c-250 ltas di$oh€d in 50 ml
slhanol (95 %). This was added to iClO ntorXmphocphoic ac6 (85
%) and vorumo was mado up ro I tirrc by gEdualy addtng u|s
slerili:od dsioniz€d watsl over a ps.iod of 3E mtn. Fitter€d twic6 wih

u7
0.45 pm n||Io c€lulo66 fll€. p€p€r (Whtmn) and ltond in e da*
bode at 4 "C.
+ Ekon. orgEn L$od
R.l0€nrr
. brtum bnsDorab (0.2 M).
. +(N, Noh.lhtamlno) b€nzald€hydo (10 g) was dBsolvod in
glad€l odc ad-10 M HCI O:li v^a rm d) and sto'ld h a
&* bd6. lmm€dbl6ly b6?s q!o, lh6 |Eag6.i we dlbt d
{1 :9) wih glaclsl acodc acu.
5. Alcortlc Add iLlhod
IL.0fnt C
Mtu(€d I volwn of6 N HFOrlih 2 \ohnEscfdbdl€d mbrand 1
volums of2.5 % ammonlum molytdab, ti€n added I volum6 of l0
% alcoftlc ac5 and mb@d $611. Thh mb(tlE we fi€6ht p|Ep€|€d
on t|e day ot u36.

?54
Arp.hdtx C
Proparalton ot Etock Eoludon cf lt .W l|dl Salt
l. C.dniun CHortd. (r )
Zol.il g CdCk tt O B difiltnd h tom nr d.htb6d ddd
2. Chrll|fur ctlortd. (l [)
206*i C crc& E didlld h to(I) d d€bod ddt€d Fbr.
3. Copp* Edph.!. (l l|,
'59.6
g ct6o.. 6ruo ft! drrDn€d in tmo d d€tonbod d.t€d
,( XtdLt 8!t at t (l I)
26?.6 I r9O.. 6tto E d!.otr€d in to(t) i{ rttao.ftsd lHsd
5. Zrcauhh.b(1 )
287.54I Z|sO.. TrlO rI dlr.otvd In tom d d.brbd d.t!€d

259
Appondix D
Preprration ot Stock Sotutlons ofAntiblo cs
i, Anplclllln
25 mg/mt in autodavd (r2r .C) disdtod war€r. SG,iriz€d by
filrrarlon (0.22 !m mittiporo fitret and slorad tn a dalk boflte €t _20
2. Chloramph.nlcol
34 mg/ml in abetut€ €th6rct. Stoled at -20 oC.
25 mg/ml in autoctav€d (121 oC) distjled v|E!sr. Slsnlz€d by
filtralion (0.22 !m miliporo tiler) and sio€d in a da* botte ar -20
"c.
20 mg/ml in auroda!€d (121 oC) disti €d mier. Sredtiz€d by
Rrtrarim (0_22 pm mi trbre tite4 and srrEd in a da.t bodo ar -20
12-5 mg/mlin absotuie €thanot. SloEd at,2O oC.

2@
Appendlx E
Stock Solutlons ot Butfers and Dyes Usgd In Agarose Gol
El6c{rophoro!ls
r. 50 r TAE (Trisac€rlie+OlA) Bufer
Tris
242 g
0.5 mM NaTEDTA (pH 8.0) too ml
Gtaci€t acetac .cid
57.1 ml
vorume was adjust€d ro t lilrc and stor€d al rcom re.nps€rule.
1 x TAE hiffs.
Oissolv€d by boiting in microwave oven
0.7 yo
100 ml
for 2 min.
ONA T6cldng Dy.
Bromophenot Btue
0.25%
40%
l0ml
Ethidium Bromlde (10 mgiht)
Ethidium Bromid€
1g
100mI
Stjrrcd untll dye h6s cornptelsty dissolvsd. StoEd In a daft bous at 4 oC.

xl
ACCanfi. F
Raagbnca !o. Daiacdng &g.r! o|| pga Chrqnatogr.tn
't. Rrg.nt A
2.5 rf editrbd sqroo! A0IO! (2 I h 4 rrt .d ffibo 16
add.d b 500 nt.o*|l.
2. R.!g.nt B
a g l&OH we. tt otv.d b senrdon h rtd .|umr d tfad
\/|Eter aod d ut d up to 200 mt $flh n or.rd.
3. Ra.Sod C
25 g N.rsrq c dbatrd h 2SZj nl d[.d *. To |l{
3ZS n ntih.m| aB dd€d.

26t
ApF.|dlI G
Stod( sd{dom md B{fit|r Gr.d ir plr!|ntd DIA Loldo|t
1. Lydrg Sotutton| (Btrnbotn dd Doty, 1929)
sobdo|| | (20() nX)
5O.r l glEo
t0
'nM
EDTA (d.t 8.0)
t.gt2 g
o.7$g
25ngT -HO0't|8,0) O.ffig
Sl€r lz.d by lubclavhg et 110 oC tur 15 .l n.nd stoEd er4t.
S.edoo ll (2OO n|)
0.2 N il€Oll
l96sDs
Pr€pdd nrdt dl d€yd'D.
Soludo.r lll (100 d)
3 M SodUm acd*
9H wo a4lrd.d b.t'8 r|tl.cdc d.
03o
2g
$.tne
z l x TE Buttbr (l0 mM tds and 1 mM EDIA)
'| I Ii€DrA (Dlro.tum E$hEo.dte.nhe tst acst tlr
X&EDTA
oLdllodwsba
O3ztg
too ml

263
pH w4 adjusl€d to 8.0 wih NaOH srjring at mqgneric slirer
r it Trt€+tct
Tns
12.14 g
100 ml
pH was a adjust€d lo 8.0 wih Hct.
rMixed 10 ml I M Tds-HCtand 1 ml 1 M NaTEDTA, and sdjustod lh6
volume to 1 tit € with Dislit€d walar oivid€d in atiquots and stedtiz€d by
autoclaving at 121 oC for Z0 min.
3.
Ly6ins Solution (Kadoand Uu relhod.
3%SDS
5 N NaOH
pH was adjust€d lo 12.6 with 2 N NaOH.
1981)
ahl
1.5 ml
0.16 ml
Mlni-Lt'rlr soluton (10 mt) (Lindqussr6r, 2006)
sDs
100 mg
0.1 ftg
0.4 ml
Rlbonucleas€ A (Sigma)
0.5 nS
1 x TAE bsfisr
Ribonuclsaso A was boited iof 10 nin as I mg/mt soturion to inactivaro
residual DilAa* aclviv_ Sotution was stored at zt oC.

M
Ae9o|dr H
Gluco.. !t.|d.d O|'v. (ph.|c|

455
Appe ft. I
Lnnurc.||c A.rd Stid.d Cuve (CJhaEb fi.O|od)
07
06
e 0.5
t0..
Eos
e02
0.1
y=0.1274
R2 | 0.9097
2g
farrrulc dd ({}

ADp.oi x J
BSA St nd.d Cu.w (Brdionl n thod)
5 6
{
I
1.!
t
0.9
0.8
0.7
03
0,5
o1
03
o2
0.1
0
Yr olrtsS.
t'. 09997
BsA(|.!)

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
267
App€ndix K
Phosphorut Standard Curve (Ascorblc acld method)
0'8
o:l
ff - 0.99s
0.6
E
€
d
0.3
s
E
0.5
0.4
o.2
0.i
PlDsplDnr (W)