REFERENCES
REFERENCES
REFERENCES
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<strong>REFERENCES</strong>
t19<br />
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Transcdptional organtz€tion ot Azotobadet lllnetedii dgcXLVtFA genas<br />
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250<br />
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:*
APPENDICES
App.ndtx A<br />
Gaouft LCa<br />
1. ll&L A.dl<br />
Nuflont brD0t (OoH)<br />
Dtddvr.br<br />
agl'.d d .l2t oC tbr 20 t|t||.<br />
2. llutld t{rr<br />
Mlttntdt(OoU)<br />
A€ir agef (Olotd)<br />
DL{lstft<br />
sbdtr.d at 12t qc ft. m |r5.<br />
,t9.0 g<br />
tom il<br />
t3.09<br />
15.0 g<br />
md<br />
3. Uft a.tlla Btrlb<br />
8.6T4fioo. (Oxoit)<br />
Btcio-Y..rt€,faot(o@ld) 59<br />
N€<br />
thfld||t|'<br />
t0 s<br />
'os<br />
io(I}m<br />
pH wa! .dlu3bd io 7.0 ulh l0 M t&OH t tb|b €i.dtdng .[ t2t .C h<br />
2() mhuaa Foa parparalioo of t-|ld*AdEj af|l ,nrrtrrlh GA agra<br />
r:dlm), t50e.rT.r(o,o5)rE d.t ,t h.tur.bbn
2:t3<br />
a. aHt<br />
B|ll 379<br />
160 n{<br />
SbrilE€d Et 121 oC fer.2o ntn, For pEparatbn of BHt e!€r medluG t5 g<br />
ag!' .gir (ol!o5) E dd.d !66.! rtdEddi.<br />
t YE r.dh|||<br />
Bo.b.Ya6toffi<br />
x&Po.<br />
trtgso4. 7!to<br />
(Nrul9O.<br />
5g<br />
Dhtiltad tN€tar<br />
20g<br />
5g<br />
3g<br />
2g<br />
lmoml<br />
pH rra! adFli.tt b 5.0 b*r€ aistadoo at t2l cC for 20 nh. SrEu<br />
frr .6.IiEd .epanbty .t fio t ft. tS fih. Fo. &Id nEdh4 t5 g<br />
agEr eger wa! edd€d b.fio|! rt$tEeton.<br />
o<br />
BAH fl.dtum<br />
B.cto.ycart oxlect<br />
N..tlPO.<br />
Cnrlc !dd. H2O<br />
Dldld ht6.<br />
20g<br />
5g<br />
5g<br />
2.7I<br />
1.15 g<br />
to(tr d
254<br />
pH was adiusi8d io 5.0 b.tore st€dttsaton ar i2t 6C. Gtuco.6 ra3<br />
sleiliz€d €spa€tety at ljo oC for t5 min. For solid mediJm. 15 g agar<br />
agar w.s add6d b€for€ stedtiza uon.<br />
Ttu{luconat. M€dtum<br />
Tds (hydoxrn€thr) aminorn6hane<br />
NaCl<br />
KCI<br />
NTLq<br />
6.06 g<br />
4.64S<br />
1.49 g<br />
1.O7 9<br />
0.439<br />
Mgclr. 6HrO<br />
CaCh_ 2H1o<br />
0.209<br />
0.039<br />
0.04 g<br />
Asar agar (Oxoid)<br />
15.0 g<br />
2.o s<br />
l0O0 ml<br />
pH wac adju3t€d to 7.0 with 2 M HCtand srgritiz€d at j21 oC for 20 min.<br />
Na. Gluconat€ viBs sradtized s€parat€ty al i i O oC for 15 min.<br />
a. KN ||odlum tor EPs pro.tE{on<br />
20g<br />
Eado-Yeast od|dct<br />
KH,PO.<br />
o5s<br />
6.8 9
l(:llFOr<br />
Nact<br />
MgaOl?HrO<br />
DirLd 6.<br />
&8C<br />
o.t c<br />
O.2g<br />
t0(tr d<br />
Elh€r glucot€ o. tiucb|€ of €{rdD!! lr€3 us€d aa cerbon &uto.<br />
Ctton ntco mr .b.t d ..pa*ty rt l tO cC ft. lO r|| e|d ura<br />
w., abo aaparably *.tb.d !t flt ton t.hg 022 lrn nttpo|! ftr.<br />
*l ms e{ded b 70 0€tbr tbdtfig d t2l t ftr 20 d.. For &ad<br />
nbdiint 'l5 C qar {fi 6 €r$sd bafta€ sffior
256<br />
Appendix B<br />
R€agents Used In Quallt ve and euen tativ. anall|.l3 of<br />
Blopolymers<br />
L Phenol-Sutturlc Actd tU€tho.l<br />
. obtiled phonoi, 5 % (w/v) in disriled w5l€i<br />
. Concontrat6d H2SO1,<br />
. Sr.ndard Stucose sotutbn (100 pSr'mD.<br />
2. Cffb.zol. M.ihod<br />
. Concedrabd HrSO. (98 ml) \NBs add€d ro sodium telraborals<br />
docahyd€t6d (0.95 g tn 2.0 mt distil€d watar) and sto€d at 40<br />
'c.<br />
. Calbazole (bcfysb insd fmm absoture €thanot; j2S rp<br />
dassoh€d in 100 mtabsotuia erhanot).<br />
3. Br.dtord ilothod<br />
cooma.ste Ro.gont (1 00 mgt)<br />
100 mg Coomassie Bdttiant Btu6 c-250 ltas di$oh€d in 50 ml<br />
slhanol (95 %). This was added to iClO ntorXmphocphoic ac6 (85<br />
%) and vorumo was mado up ro I tirrc by gEdualy addtng u|s<br />
slerili:od dsioniz€d watsl over a ps.iod of 3E mtn. Fitter€d twic6 wih
u7<br />
0.45 pm n||Io c€lulo66 fll€. p€p€r (Whtmn) and ltond in e da*<br />
bode at 4 "C.<br />
+ Ekon. orgEn L$od<br />
R.l0€nrr<br />
. brtum bnsDorab (0.2 M).<br />
. +(N, Noh.lhtamlno) b€nzald€hydo (10 g) was dBsolvod in<br />
glad€l odc ad-10 M HCI O:li v^a rm d) and sto'ld h a<br />
&* bd6. lmm€dbl6ly b6?s q!o, lh6 |Eag6.i we dlbt d<br />
{1 :9) wih glaclsl acodc acu.<br />
5. Alcortlc Add iLlhod<br />
IL.0fnt C<br />
Mtu(€d I volwn of6 N HFOrlih 2 \ohnEscfdbdl€d mbrand 1<br />
volums of2.5 % ammonlum molytdab, ti€n added I volum6 of l0<br />
% alcoftlc ac5 and mb@d $611. Thh mb(tlE we fi€6ht p|Ep€|€d<br />
on t|e day ot u36.
?54<br />
Arp.hdtx C<br />
Proparalton ot Etock Eoludon cf lt .W l|dl Salt<br />
l. C.dniun CHortd. (r )<br />
Zol.il g CdCk tt O B difiltnd h tom nr d.htb6d ddd<br />
2. Chrll|fur ctlortd. (l [)<br />
206*i C crc& E didlld h to(I) d d€bod ddt€d Fbr.<br />
3. Copp* Edph.!. (l l|,<br />
'59.6<br />
g ct6o.. 6ruo ft! drrDn€d in tmo d d€tonbod d.t€d<br />
,( XtdLt 8!t at t (l I)<br />
26?.6 I r9O.. 6tto E d!.otr€d in to(t) i{ rttao.ftsd lHsd<br />
5. Zrcauhh.b(1 )<br />
287.54I Z|sO.. TrlO rI dlr.otvd In tom d d.brbd d.t!€d
259<br />
Appondix D<br />
Preprration ot Stock Sotutlons ofAntiblo cs<br />
i, Anplclllln<br />
25 mg/mt in autodavd (r2r .C) disdtod war€r. SG,iriz€d by<br />
filrrarlon (0.22 !m mittiporo fitret and slorad tn a dalk boflte €t _20<br />
2. Chloramph.nlcol<br />
34 mg/ml in abetut€ €th6rct. Stoled at -20 oC.<br />
25 mg/ml in autoctav€d (121 oC) distjled v|E!sr. Slsnlz€d by<br />
filtralion (0.22 !m miliporo tiler) and sio€d in a da* botte ar -20<br />
"c.<br />
20 mg/ml in auroda!€d (121 oC) disti €d mier. Sredtiz€d by<br />
Rrtrarim (0_22 pm mi trbre tite4 and srrEd in a da.t bodo ar -20<br />
12-5 mg/mlin absotuie €thanot. SloEd at,2O oC.
2@<br />
Appendlx E<br />
Stock Solutlons ot Butfers and Dyes Usgd In Agarose Gol<br />
El6c{rophoro!ls<br />
r. 50 r TAE (Trisac€rlie+OlA) Bufer<br />
Tris<br />
242 g<br />
0.5 mM NaTEDTA (pH 8.0) too ml<br />
Gtaci€t acetac .cid<br />
57.1 ml<br />
vorume was adjust€d ro t lilrc and stor€d al rcom re.nps€rule.<br />
1 x TAE hiffs.<br />
Oissolv€d by boiting in microwave oven<br />
0.7 yo<br />
100 ml<br />
for 2 min.<br />
ONA T6cldng Dy.<br />
Bromophenot Btue<br />
0.25%<br />
40%<br />
l0ml<br />
Ethidium Bromlde (10 mgiht)<br />
Ethidium Bromid€<br />
1g<br />
100mI<br />
Stjrrcd untll dye h6s cornptelsty dissolvsd. StoEd In a daft bous at 4 oC.
xl<br />
ACCanfi. F<br />
Raagbnca !o. Daiacdng &g.r! o|| pga Chrqnatogr.tn<br />
't. Rrg.nt A<br />
2.5 rf editrbd sqroo! A0IO! (2 I h 4 rrt .d ffibo 16<br />
add.d b 500 nt.o*|l.<br />
2. R.!g.nt B<br />
a g l&OH we. tt otv.d b senrdon h rtd .|umr d tfad<br />
\/|Eter aod d ut d up to 200 mt $flh n or.rd.<br />
3. Ra.Sod C<br />
25 g N.rsrq c dbatrd h 2SZj nl d[.d *. To |l{<br />
3ZS n ntih.m| aB dd€d.
26t<br />
ApF.|dlI G<br />
Stod( sd{dom md B{fit|r Gr.d ir plr!|ntd DIA Loldo|t<br />
1. Lydrg Sotutton| (Btrnbotn dd Doty, 1929)<br />
sobdo|| | (20() nX)<br />
5O.r l glEo<br />
t0<br />
'nM<br />
EDTA (d.t 8.0)<br />
t.gt2 g<br />
o.7$g<br />
25ngT -HO0't|8,0) O.ffig<br />
Sl€r lz.d by lubclavhg et 110 oC tur 15 .l n.nd stoEd er4t.<br />
S.edoo ll (2OO n|)<br />
0.2 N il€Oll<br />
l96sDs<br />
Pr€pdd nrdt dl d€yd'D.<br />
Soludo.r lll (100 d)<br />
3 M SodUm acd*<br />
9H wo a4lrd.d b.t'8 r|tl.cdc d.<br />
03o<br />
2g<br />
$.tne<br />
z l x TE Buttbr (l0 mM tds and 1 mM EDIA)<br />
'| I Ii€DrA (Dlro.tum E$hEo.dte.nhe tst acst tlr<br />
X&EDTA<br />
oLdllodwsba<br />
O3ztg<br />
too ml
263<br />
pH w4 adjusl€d to 8.0 wih NaOH srjring at mqgneric slirer<br />
r it Trt€+tct<br />
Tns<br />
12.14 g<br />
100 ml<br />
pH was a adjust€d lo 8.0 wih Hct.<br />
rMixed 10 ml I M Tds-HCtand 1 ml 1 M NaTEDTA, and sdjustod lh6<br />
volume to 1 tit € with Dislit€d walar oivid€d in atiquots and stedtiz€d by<br />
autoclaving at 121 oC for Z0 min.<br />
3.<br />
Ly6ins Solution (Kadoand Uu relhod.<br />
3%SDS<br />
5 N NaOH<br />
pH was adjust€d lo 12.6 with 2 N NaOH.<br />
1981)<br />
ahl<br />
1.5 ml<br />
0.16 ml<br />
Mlni-Lt'rlr soluton (10 mt) (Lindqussr6r, 2006)<br />
sDs<br />
100 mg<br />
0.1 ftg<br />
0.4 ml<br />
Rlbonucleas€ A (Sigma)<br />
0.5 nS<br />
1 x TAE bsfisr<br />
Ribonuclsaso A was boited iof 10 nin as I mg/mt soturion to inactivaro<br />
residual DilAa* aclviv_ Sotution was stored at zt oC.
M<br />
Ae9o|dr H<br />
Gluco.. !t.|d.d O|'v. (ph.|c|
455<br />
Appe ft. I<br />
Lnnurc.||c A.rd Stid.d Cuve (CJhaEb fi.O|od)<br />
07<br />
06<br />
e 0.5<br />
t0..<br />
Eos<br />
e02<br />
0.1<br />
y=0.1274<br />
R2 | 0.9097<br />
2g<br />
farrrulc dd ({}
ADp.oi x J<br />
BSA St nd.d Cu.w (Brdionl n thod)<br />
5 6<br />
{<br />
I<br />
1.!<br />
t<br />
0.9<br />
0.8<br />
0.7<br />
03<br />
0,5<br />
o1<br />
03<br />
o2<br />
0.1<br />
0<br />
Yr olrtsS.<br />
t'. 09997<br />
BsA(|.!)
0<br />
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9<br />
267<br />
App€ndix K<br />
Phosphorut Standard Curve (Ascorblc acld method)<br />
0'8<br />
o:l<br />
ff - 0.99s<br />
0.6<br />
E<br />
€<br />
d<br />
0.3<br />
s<br />
E<br />
0.5<br />
0.4<br />
o.2<br />
0.i<br />
PlDsplDnr (W)