Il

Muterials dttd Melhols 33

,{i ,}..
\-,1 Smattscalepreparation
of theplasmidDNA

isolationof plasmidDNA was carriedout by alkali lysis methodof

r Small-scale

, Brinboim and Dolly ( I 979), as described_

by Sambrooket al. (l 989).

A single isolatedcolony was inoculatedin 3 ml Luria broth (LB) medium

continuous
shaking
nt 250fpm in 0n incubalor (NewBrunswick
shaker Inc,

\ew Jirs\').USAr.

2. 1.5ml ofthe overnight

grownculturewastransferred
to a micro-centrifuge

tubeandcellswerepelleted
(13000
rpm,I min).

i. l'he supernatant
wasdiscarded in 100pl ice-cold
andcellsweresuspended

CTE buffer(0.05M clucose,0.025

M TrisCI,pH 8,0and0.01M EDTA,

pH 8.0)by vortexing,

4, 200 ;tl freshlyprepafed (l% SDS and 0.2 N NaOII) was

alkaline-SDS

a(ldedandthc cellswefelvsedby gentl),invertingthe tubesevefal

times,
'l'helysatcrvaskeptat foomtemperatufe
lof 5 min,

5. 150prl of ice-coldpotassium solution(3 M potassium

acerate and5 M

acetate)
wasaddedandthe contents
weremixedgently.Now the lysatewas

kepton icefor I0-l5 min.

6. Thetlrbecontaining
the lysatewascentrifuged
(13,000rpm, l5 min,4"C).

The supernatant
was tmnsferred
to a ftesheppendorftube,extractedonce

JI,IATERIAL
AND METHOD 33
 Materials und Methods 34

withequalvolumeof phenol:chloroform: iso.amyl

alcohol(25:24:lv/v)and
t\rrcewith chloroform: iso-amylalcohol(24:l).

7. The nucleicac;dsin aqueousphasewere precipitated

with 2 volumgsof
ethanolbJ incubating
at _200Cfor 20 min.
8. fhe nuclejcacidsqere pellered by cenlrilugarion
{13.000rpm. l5 min,
4"C),thepelletwaswashedwith iceoold70%
ethanolanddriedin air.
9. The nucleicacidpelletwasdissolved
in 50 pl TrisCl(10 mM, pH 8.0)
containingDNaselree RNaseA(20 pglml) and
incubatedat 37oCfor I h.
The plasmidDNA was extractedwith chlorofbrm:isoamyl
alcoholand
precipitated
asdescribed
earlierin steps
7_g.
I0. Theplasmid
DNA wasdissolved
in 50pl water.3plofsamplewaschecked
on 0.8olo
agarose
gel.TheplasmidDNA wasstoredat _20oC,

q, 6, g - Ligation
ofpcR productin ctoning
vector

tl|fi"9 PCRproduct(insertDNA) arld_plallniq

DNA (vectorDNA, SK*/.)was

!1rT{ggr I% and 30%aghrose

gel with markerat knownconcentration
--'=' and
speckophotometer
Tlill:gl DNA was cloned-into _EcoRV
sitesof rhe vector
D-NA 150 ng of the digestedvectorandappropfiate
amountof insef DNA in a
molarratioof l:3 weremixedandligationmixture
wasmadeasfollows:_

Vector = 150ng
rnseft = 3X in mo]arratio

I igarion
buflerr l0xr I gl

IIUTERIALANDMETHOD
34
 Materials and Methods 36

this platewas inoculatedto 5 ml LB mediumand grown (37"C, 200 rpm, l0 h). 100

mlofLB medium (in lL flask) was inoculatedwith l00pl ofthis cultureand grown

to an OD of 0.3 to 0.4 at 600 nm by vigorous shaking (300 rpm, 37'C). The

bacterial
cellswerepelleted
in SS34tube(2700xg,
5 min, 4"C).Afterthisall the

stepswereconducted
on ice.Thepelletwasgentlysuspended
(till no olumpswere

left) in 20 ml ice-cold0.1 M CaCl2and kept on ice for 30 min. The cells were

pelletedagain(asin prcviousstep),resuspended
in 20 ml ice,cold0.1M MgCll and

kepton ice for 30 min.The coli cellswerecentrifuged

andsuspendedin 4 ml of
',
ice-cold0.1M CaCl2containingl0% glycerolanddispensed of200 pl
in aliquots

in 1,5ml sterileeppendorftubes.
Thecellswerestoredat-70"C till fu(h,e^[
use.

L1,6,1 ;- Ttansto.-otiohof competent

E colicclls

PlasmidDNA or ligated/mixtufe
\.!qgmixedgentlyto 200 pl o-f*comp€tent

c,ellsandkepton ice for 30 min.A briefheatshock(90 seconds)

at 42oCwasgiven

andthe tubeswerer.eplaced
cinice for 2 ntin. 800lLl LB mediumwasaddedto the

cellsuspcnsion
andincubated (200rym,45nin! 37"C).200pl bacrerial
oDshakef

suspension
was platedon LBA platescontainingthe appropriateantibiotic.The

plateswere incubated
overnightat 37oC.C,olonies
growingon the antibioticplate

wgg.91Ec!9d
fof recqmbinant
plasmid.
\

Selectiorof recombinant,. cd,1j

by o complemcntttion

M4TER]ALAND METHOD 36
 Malerials and Metlrc.ls 37

Using a sterileglassspreader,40 pl of stocksolutionofX-Gal (20 mg/ml in

dimethylformamide)and 8 pl of IPTG (200 mg/ml in H2O) were spreadon LBA

platescontainingampicillin antibiotic(150 pglml) and left for drying for about30

min. 200 pl oI thc transfonnedcells was uniformly spreadon these plates.The

plateswere incubatedat 37.C ovemight,The white_colonieswere--streaked

on the
maslerplateand analyzedfor recombinantplasmidsubsequently.
t/
t/"
L , 6,l) . ] Scleclion
ofE colihrvingrccom
binant
plasm
id nr.fr".i", Oifr"*"i;;i
\,/l-> "f
DNA frog enrs

While coloniesor
of z.
E. col/
coli were
wercfurther
lurthercheckedior plasmid,
for recombinant
plasmid
:T.-col:nrcs
The plasmidDNA was isolatedfrom severalputativerecombinant E. coli cellsr
*foHowlng-rnin.iprep-grotocol.
The plasmid DNA was analyzedby restriction

digestionor colony pCR. Severalclonesrepresenting

fragmentsof the activator
motifswercsequenced
on autoniated
DNA sequencer
(Bangalore
genel).
. c . ( o r\
ir\' L "' ;' 1 ' )
,. ! rt.
Y. o ' / r l
t,t.ct':rrarioI I'rol]e:_
ofnladio_taE]gy
L{'{r.l!- >--'
-'5ar;ii'red theDNA remplarebyhearing
, , ,, ,. it in a microc(ntrifuge
tubefor l0min. at
-4,ir.l\'1 ,
95-1000C.Chilled the tube rapidly in an ice bath. To generatethe DNA probe,

preparedthe following reactionmixture:

I
I
DNA template 500ngoptimum
I X enzymebuffer 2 gl

IATER]AL AND METHOD 37

 Materisls dnd Methods 38

(CTP,ATPandTTP)
Deoxynucleotides 20 pM (Each).

la]'?Pl crP 3ooiM(3,ooo

ci/mmo)
:-"
Klenowfragment L0 rrl(3U)

thereactionfor I hrsat 370C.Stopped

Incubate thereactionafteradding0.5M

EDTA (20 mM final concentration)

to themixtureor by heatingthe mixturefor l0

min.at 750C.

1 Ol i e oD T Me th o d-:
1 ,1. b, , l sr ,.. tr - ,l :
.
I'
L Prepared
mRNA(2-5)andmixedwitholigo(dt)
primerandmadethefinal

vo l u mel 5 p l .

2. Themixturewasheatedat 700Cfor l0 min.andthetubewaschilledon icefor 5

't$
min.andspinnedbrieflyto colleotthesolution.

3. Thefollowingcomponent
wereadded:

Sample 15pl

F i rststfa n d 5 X b u fl e r 5pl

RNasin*
Ribonuclease
inhibitor l 0 pl (40U)

20mMsoiution
ofdGTP,dATP& dTTP 2pl

125pM dCTP 2 ttl

o" Pp.CTP(3,000Ci/mmole) 2pl

AMV-Reverse
Transcriptase L0 pl (J0U)

RNasefree Water 27.0 1tl

MATERIALAND METHOD 38