Basic Molecular Biology & Biotechnology Training Manual

I. The Molecular Design of Life 1. Prelude: Biochemistry and the Genomic Revolution 1.1. D ! Illustrates the Relation "et#een $orm and $unction

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Contents
S No Name of chapter
1. List o1 *nstruments

Page No " 4 . / 6+2 1rom Bacteria %$ %" %4 %%# %/ %: "$ "% 7

"(

2bbre3iations

%. Preparation o1 Bu11ers 5 Solutions &. Bacterial Culture '. *solation o1 Plasmi

(. *solation o1 Genomic 6+2 1rom Bacteria ). *solation o1 Genomic 6+2 1rom Bloo *. 2garose Gel 8lectrophoresis +. 9uanti1ication o1 6+2 1,. 11. 1-. 1%. 1&.

Restriction igestion o1 6+2 Ligation o1 6+2 1ragments Polymerase Chain Reaction 8lution o1 6+2 1rom 2garose Gels S6S P2G8

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1 - List of Instruments
%( "( 4( .( -( #( /( :( 7( +on Re1rigerate Centri1uge Re1rigerate Centri1uge Balance B;6 *ncubator <ree=er Gel 8lectrophoresis 'nit >icropipettes ?orte@ 6ry Bath

%$( Aater Bath %%( *ncubator Shaker %"( '? Bransilluminator %4( Gel 6ocumentation System %.( Aater Puri1ication System %-( Programmable Bhermal Cycler %#( Laminar 2ir <low %/( Gel Rocker
1*.

>agnetic Stirrer

%7( Cot Plate "$( Spectrophotometer "%( pC >eter ""( ?arious Glasswares 5 Plasticwares

2-

!!re"iations & Sym!ols use#

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6A RB g m> > + ml Dl S6S 86B2 2PS CBB LB BPB B8 nm '? , mol( wt 8tBr ? cm bp kb ;6

, , , , , , , , , , , , , , , , , , , , ,

6istille water Room temperature gram mili molar >olar solution +ormal solution mili liter micro liter So ium 6o ecyl Sulphate 8thylene 6iamine Betra 2cetate 2mmonium Persulphate Commassie Brilliant Blue Luria Bertani Bromo Phenol Blue Bris 86B2 , nano meter ultra 3iolet molecular weight 8thi ium Bromi e 3olts , centi meter base pairs kilo base optical ensity

$ - Preparation of Buffers & Solutions

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Solution I
a) b) c) ) Glucose Bris-Cl &pC :($) 86B2 &pC :($) 2utocla3e an store at .($EC -$m> "-m> %$m>

Solution II a) +a;C b) S6S $("+ %F

Solution III %for &'ml( or Potassium

cetate

%$M(
a) -> Potassium 2cetate b) Glacial 2cetic 2ci c) 6A Cell Lysis Buffer a) %$m> Bris &pCG:($) b) %m> 86B2 c) $(%F S6S &wH3) 4$ml -(/-ml %.("-ml

)el loa#ing #ye

a( !( c( d. BPB ,ylene Cyanol Sucrose Store at .EC '*2&+ '*2&+ -'*'+

T. %1',(
a( Tris-Cl %p/01*'( !( .2T %p/01*'( 1''mM 1'mM

NaCl %&M(

To prepare &M solution #issol"e 232g of NaCl in 1''ml of 24* #5ust the "olume to 1 liter 6ith 24* 2ispense in ali7uots8 autocla"e an# store at 9T*

So#ium

cetate %$M(

2issol"e -'1*$g of So#ium cetate in 1''ml 24* #5ust p/ to &*2 6ith )lacial cetic ci#* #5ust the "olume to 1 liter 6ith 24* 2ispense in ali7uots an# sterili:e !y autocla"ing*

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.thi#ium Bromi#e
1''mg .tBr is #issol"e# %6ith the help magnetic stirer(in 1'ml 24 an# store# in #ar; !ottle at RB(

'*&M .2T

%p/ 1*'(

2 %:#(%g o1 6iso ium 8thylene 6iamine Betra 2cetate in :$$ml 6A( Stir 3igorously on a magnetic stirrer( 2 Iust pC to :($ with +a;C( >ake up to % liter by 6A( 2utocla3e an store at RB(

T . %&',(
6issol3e "."g o1 Bris in 6A( 2 -/(%ml o1 Glacial 2cetic 2ci an a %$$ml o1 $(-> 86B2 &pCG:)( >ake up to % liter by 6A( 2utocla3e an store at RB( 9esol"ing )el< 1&ml8 12*&+ a. ". c. d. e. f. 6A #(#$4ml Resol3ing gel bu11er %(:/-ml 2crylami eHbis-acrylami e &4$,$(:) :(44ml %$F S6S %$$Dl %$F 2PS %$$Dl B8>86 ""Dl

9esol"ing )el Buffer %1*&M Tris8 p/ 1*1( 6issol3e %:(%-g Bris base in /$ml 6A( 2 Iust pC with %+ CCl( >ake up to %$$ml by 6A an store at RB( Stac;ing )el< &ml8 &+ a. b) c. ) e) 1) 6A "(#".ml Stacking gel bu11er %("-ml 2crylami eHbis-acrylami e&4$,$(:) %($ml %$F S6S -$Dl %$F 2PS -$Dl B8>86 %%Dl

Stac;ing )el Buffer %1*'M Tris8 p/ =*1( 6issol3e %"(%g Bris base in /$ml 6A( 2 Iust pC with %+ CCl( >ake up to %$$ml by 6A an store at RB( Tris )lycine .lectro#e Buffer %p/ 1*$( a) Bris 4($g

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b) Glycine %.(.g c) 6issol3e in :$$ml 6A, a Iust pC to :(4, make up to %$$$ml an store at RB

crylami#e>!is- crylami#e Solution

a) 2crylami e 4$g ". Bis-acrylami e $(:g c) 6issol3e in 6A, make up to %$$ml an store at .EC

mmonium Persulphate Solution %1'+(

6issol3e %$$mg o1 2PS in %ml 6A an Prepare 1resh solution e3ery time( store at .EC(

S2S Solution %1'+(

6issol3e %$g S6S in %$$ml 6A an store at RB(

)el Staining Solution

a) Commasie Brilliant Blue b) >ethanol c) 2cetic 2ci ) 6A <irst issol3e CBB in methanol an water then a $("-F .-F %$F .-F 2cetic 2ci (

2estaining Solution
a) >ethanol b) 2cetic 2ci c) 6A a. b) c) d. e. 1) .-F %$F .-F

Sample

Preparation Bu11er

$(#> Bris-Cl &pCG#(:) -ml S6S $(-g Sucrose -($g /->ercaptoethanol $("-ml %F Bromophenol Blue "(-ml 2 6A to make up to -$ml an store at RB

#J Gel Loa ing 6ye a) Bromophenol Blue &wH3) b) Jylene Cyanol $("-F &wH3) $("-F

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Sucrose &wH3) Store at .oC

c.

.$($$F

- - Bacterial Culture
2 culture o1 bacteria &E.coli) is normally recei3e as a broth culture, on a Petri ish or as a 1ree=e- rie culture( <irst o1 all a care1ul recor o1 the strain no an genotype o1 the strain is maintaine ( By this, one can easily i enti1y an appropriate me ium an any a itions such as antibiotics, which are necessary to ensure stability an maintenance o1 plasmi s( *t is necessary to ry the agar plates because E.coli is motile an it swims across the plate in the thin 1ilm o1 water, moreo3er contaminants also sprea easily across the plate an esire single colonies cannot be isolate ( Bo achie3e this ry the plates o3ernight at 4/EC in an o3en(

Protocol 1or preparation o1 2gar Plates

a. Aeigh .F LB agar me ium in a conical 1lask( Pour the esire Kuantity o1 6A an mi@ it by swirling the 1lask( b) Boil in a microwa3e o3en( Swirl the liKui to check that the agar is 1ully melte ( Bake care that agar oes not boil o3er( c. 2utocla3e an allow the me ium to cool up to .---$EC( 2 appropriate conc o1 antibiotic, stir to mi@ well an allow time 1or any air bubble to isappear( ) 2rrange the sterile Petri plates on a le3el sur1ace an label the base o1 each plate to in icate the me ium prepare (

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e) <lame the neck o1 the 1lask containing me ium an pour the reKuire amount &appro@imately %$ml 1or short term bacterial culture) o1 me ium into the plates( f. 2llow the plates to set( 6ry the sur1ace o1 the plates by o3ernight incubation at 4/EC( Check 1or contamination ne@t morning an iscar the plates with contamination( g. Arap the plates with para1ilm an store the plates at .EC(

Single colony isolation

Bhe principle o1 this techniKue is to streak a suspension o1 bacteria until single cells are separate on the plate( 8ach in i3i ual cell then grows in isolation to pro uce a clone o1 i entical cells known as a LColony(M Bhe maIority o1 these cells are genetically i entical( Cowe3er, uring growth, mutation o1 e3en a single colony can gi3e rise to low le3els o1 mutant cells( Repeate single colony isolations result in a pure culture(

Protocol &streak plate metho )

a) <lame a nichrome loop &4mm across an has #cm stem)( 2llow the loop to cool or cool by immersion in a sterile area o1 the me ium( b) <lame the neck o1 an o3ernight LB Broth culture an remo3e a loop o1 cells or pick a colony 1rom o3ernight LB 2gar culture( c. Streak the cells at one si e o1 a well- rie agar plate &in a manner to make the 1irst arm o1 a pentagon)( Streak se3eral times close together( ) <lame the loop an cool as be1ore( e) Streak again, starting 1rom one en to make secon arm o1 pentagon( 1) Repeat the steps ) an e) to complete the pentagon( g. Label each plate to in icate the strain no, genotype o1 strain, ate o1 culture an name o1 person( h) Arap the plates with para1ilm( i. *ncubate the plate at 4/EC with the me ium 1acing ownwar s to re uce the chance o1 roplets o1 con ensation 1alling on the me ium sur1ace( 8@amine the plates 1or colony morphology an the presence o1 possible contaminants( *1 all o1 the colonies are o1 uni1orm si=e an appearance it is assume to be a pure culture( Subculture o1 almost any colony shoul gi3e the reKuire strain an the plate is worth keeping as a 1uture source o1 a puri1ie culture(

Broth culture Protocol

a. Prepare %(-F o1 LB broth me ium in a conical 1lask( ". 2utocla3e an allow the me ium to cool up to .---$EC( 2 appropriate conc o1 antibiotic, stir to mi@ well, allow time 1or any air bubble to isappear(

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c. 2rrange the sterile test tubes an label each test tube to in icate the strain no, genotype o1 strain, ate o1 culture an name o1 person( d. <lame the neck o1 the 1lask containing me ium an pour the reKuire amount &appro@imately .--ml ) o1 me ium into the test tubes an 1i@ cotton plug e) Pick a single colony by a sterile toothpick 1rom agar plates an culture the test tube( 1) Repeat step e) 1or as many test tubes as reKuire ( g. Place the tubes in an incubator shaker at 4/EC, "-$rpm o3ernight(

- - *solation o1 Genomic 6+2 1rom Bacteria

*ntro uction
*solation o1 intact high molecular weight 6+2 with su11icient purity is a basic reKuirement 1or any genomic stu y( *n 1act, the application o1 molecular biology techniKues to analy=e genome epen s on the ability to isolate pure, high molecular weight 6+2( 2 protocol that works 1or a group may 1ail with others( ConseKuently, a number o1 6+2 isolation metho s ha3e been e3elope 1or i11erent target groups( Cence, protocol selecte shoul be a eKuate, Kuick, simple, an cost e11ecti3e an it shoul yiel 6+2 reasonably pure an intact( Bhe two 1actors that a11ect the integrity o1 6+2 are mechanical shearing an nuclease acti3ity( Bhe 1ormer can be taken care by a3oi ing harsh treatment o1 lysates an taking measures to inacti3ate the en ogenous nuclease acti3ity can control the latter( Bacteria are the simplest material to process an can easily be har3este by centri1ugation( 6uring the isolation o1 6+2 1rom

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Bacteria the lysis bu11er containing +a;C an S6S, Proteinase K is use 1or the remo3al o1 the R+ase acti3ity or e@tra R+2 present in the e@tract( Phenol is 3ery goo sol3ent o1 proteins but can also issol3e small Kuantities o1 6+2, 1or this reason, it is pre1erable to use phenol chloro1orm on 6+2 sample which are not hea3ily contaminate because 6+2 oes not issol3e in this organic mi@ture( 6+2 is puri1ie an concentrate with ethanol an isopropanol( So ium 2cetate with S6S 1orms the network o1 the contaminating agents such as membranes, high mol wt R+2, proteins(

Reagents
a) b) c) ) e) 1) %$F S6S &wH3) "$mgHml Proteinase K Phenol,chloro1orm *sopropanol 4> So ium 2cetate B8

Protocol
a) Bake %(-ml o1 bacterial culture in a micro1uge tube an spin at %$$$$rpm 1or 4 min( ". 6ecant the supernatant an resuspen the pellet in .#/Dl B8 bu11er by repeate pipetting( c. 2 4$Dl o1 %$F S6S an 4Dl o1 "$mgHml Proteinase K, mi@ well an incubate 1or " hrs at 4/EC( d. 2 an eKual 3olume o1 Phenol,chloro1orm an mi@ well by in3erting, until the phases are complete mi@e ( e) Spin at %$$$$rpm 1or - min an trans1er the upper aKueous phase to a new tube( 1) Repeat the step ) an e) 1or once( g) 2 %H%$ 3olume o1 4> so ium 2cetate an $(- 3olume o1 *sopropanol( >i@ gently until the 6+2 precipitates( h) Centri1uge at %$$$$rpm 1or - min( 6iscar the supernatant care1ully an air- ry the pellete( i. Resuspen the pellet in %$$Dl B8 bu11er( 0. Store at -"$EC(

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# - *solation o1 Plasmi 6+2 1rom Bacteria

*ntro uction
Plasmi isolation is achie3e by the isruption o1 cell wall by alkali lysis( Bacteria are suspen e in an isotonic solution o1 sucrose an a1ter a ition o1 86B2, the cells are e@pose to etergent an lyse by treatment with alkali( Bhe treatments, which isrupt base pairing, cause the linear chromosomal 6+2 o1 the host to enature( Cowe3er, the stran s o1 close circular plasmi 6+2 are unable to separate 1rom one another because they are topologically interwine ( Ahen con itions &pC) are returne to normal, the stran s o1 the plasmi 6+2 rapi ly 1all into per1ect register an completely nati3e super helical molecules are re1orme ( But, chromosomal 6+2 can not re-nature an 1orms a network consisting o1 chromosomal 6+2Hhigh mol( wt( R+2HS6SHProteinsHmembrane

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comple@es, which can be remo3e by centri1ugation( Plasmi 6+2 is puri1ie an concentrate with ethanolHisopropyl alchohol(

9eagents Protocol
a) Prepare a culture o1 bacteria containing the plasmi with su11icient aeration an the appropriate antibiotic( ". Brans1er %(-ml o1 the culture to a micro1uge tube an centri1uge at %"$$$rpm 1or % min at .EC to 1orm a pellet( c. 6ecant the supernatant, an resuspen the cell pellet in icecol $("ml solution *( 8nsure that a homogeneous suspension is 1orme ( d. 2 $(4ml 1reshly prepare solution ** an in3ert the cappe tube se3eral times to mi@ the contents( Bhe solution shoul be translucent an 3iscous( Store the tube on ice 1or - min( e. 2 $("-ml o1 ice-col solution ***( >i@ the contents by in3erting the tube se3eral times( Store the tube on ice 1or min( f. Centri1uge the bacterial lysate at %"$$$rpm 1or - min at .EC( Brans1er the supernatant to a 1resh tube( g. 2 an eKual 3olume o1 phenol,chloro1orm( >i@ the organic an aKueous phases, an then centri1uge the emulsion at %"$$$rpm 1or " min at .EC( Brans1er the upper aKueous phase to a new tube( h. Precipitate the plasmi 6+2 1rom the supernatant by a ing " 3olumes o1 ethanol at RB( >i@ an lea3e the tube at RB 1or 4 min( i. Centri1uge at %"$$$rpm 1or - min at .EC( I) 6iscar the supernatant gently, an in3ert the tube on tissue paper( 1. 2 %ml o1 /$F ethanol to the pellet an in3ert the tube se3eral times( l. Centri1uge at %"$$$rpm 1or 4 min at .EC( m. 6iscar the supernatant an air ry the pellet( n. 6issol3e the pellet in %$$Dl o1 B8 &pC :($) containing "$DgHml 6+ase-1ree R+ase( >i@ the solution gently( o. Store at -"$EC(

/ - *solation o1 Genomic 6+2 1rom Bloo

*ntro uction

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Pro uction o1 great bio banks an 6+2 banks in national 5 international le3els is one o1 e3elopment pi3ots, which is 3ery important in me ical, agricultural, economical 5 1orensic genetic 1iel s( *n this regar , one o1 the primary an important steps 1or all is 6+2 e@traction with high Kuality 5 Kuantity in minimum time 1rom cells( By using the 1ollowing metho , genomic 6+2 with high Kuality 5 Kuantity can be acKuire in the shortest time( 6+2 prepare 1rom bloo as escribe in this protocol is suitable 1or use as a template in PCRs(

Reagents
a) b) c) ) e) 1) g) h) i) 8thanol *sopropanol Potassium 2cetate &->) Cell lysis bu11er B8 &pCG/(#) Bris-Cl "$m> &pCG/(#) 6+ase 1ree R+ase &.mgHml) Proteinase K &"$mgHml) Bloo

Protocol
a. Brans1er 4$$Dl o1 whole bloo into two >CB( b) 2 7$$Dl o1 "$m> Bris-Cl &pCG/(#) to each tube an in3ert the cappe tubes to mi@ the contents( *ncubate the tubes at RB 1or %$min, occasionally in3erting the tubes( c) Centri1uge the tubes at %.-$$ rpm 1or %min at RB( ) 6iscar all but "$Dl o1 each supernatant( e. Resuspen the pellets o1 white cells in small amount o1 supernatant le1t in each tube an repeat once 1rom step b( 1) Combine the resuspen e cell pellet in a single tube an a #$$Dl o1 ice-col cell lysis bu11er( Comogeni=e the suspension Kuickly( &Bhe S6S will precipitate 1rom the ice-col cell lysis bu11er pro ucing a clou y suspension) g. 2 4Dl o1 Proteinase K solution to the lysate an incubate the igest 1or at least 4 hrs at --oC( h. 2llow it to cool to RB an a 4Dl o1 6+ase 1ree R+ase( *ncubate the igest 1or 4$min at 4/oC( i. 2llow the sample to cool to RB( 2 "$$Dl Potassium 2cetate solution an mi@ the contents o1 the tube by 3orte@ing 3igorously 1or "$sec( 0. Centri1uge to pellet the proteinHS6S comple@ at %.-$$rpm 1or 4min at .oC( &*1 the pellet is not 3isible, incubate the lysate 1or -min on ice an repeat the centri1ugation step)( 1. Brans1er the supernatant to a 1resh >CB an a #$$Dl *sopropanol( >i@ the solution gently( l) Reco3er the precipitate o1 6+2 by centri1uging the tube at %.-$$rpm 1or %(-min at RB(

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m) 6iscar supernatant an a #$$Dl o1 /$F 8thanol to the 6+2 pellet( *n3ert the tube se3eral times an centri1uge the tube at %.-$$rpm 1or %min at RB( n) Care1ully iscar supernatant an allow the 6+2 pellet to ry in air 1or "$min( o. 6issol3e the pellet o1 6+2 in %$$Dl o1 B8 &pCG/(#)( Bo 1acilitate solubili=ation o1 6+2 pellet incubate at --oC 1or 4$min(

: - 2garose Gel 8lectrophoresis

*ntro uction
Bhis techniKue is repeate ly use in molecular biology to separate 6+2 1ragments an to assess Kuality an Kuantity o1 6+2 samples(

Principle
Bhe gel is ma e 1rom 2garose, which is a copolymer o1 6galactose an 4,#-anhy ro-L-galactose( *t 1orms a gel by hy rogen bon ing an the pore si=e o1 gel epen s on the concentration o1 agarose in the gel( Low agarose concentration impro3es resolution o1 larger 1ragments but re uces resolution o1 smaller 1ragments an 3ice 3ersa( Cence, concentration o1 agarose in the gel is eci e accor ing to the si=e range o1 6+2 species to be separate ( Bhe gel is immerse in bu11er an the 6+2 samples are loa e onto a well at one en o1 the gel an ma e to mo3e through the gel by the application o1 electric current( Since, 6+2 is negati3ely charge hence will mo3e towar s the ano e( Cowe3er, the polysacchari e mi@ o1 the gel retar s mo3ement o1 the 6+2 by a process o1 sie3ing, so that small 1ragments mo3e through 1aster an these 1ragments separate accor ing to si=e( Bhe 6+2 samples are 3isuali=e by a ing 8tBr, a 1luorescent molecule which intercalates within the 6+2 bases, e@ten ing the

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length o1 linear an nicke 6+2 molecules an making them more rigi ( Ahen 8tBr is a e , '? ra iation at "-.nm it is absorbe by the 6+2 an transmitte to the boun ye( Bhe energy is re-emitte at -7$nm in the re -orange region o1 the spectrum( 8tBr is a power1ul mutagen an hence the gel shoul be han le care1ully with the glo3es( Bhe 6+2 ban s can be 3isuali=e un er '? an the ata can be recor e by gel ocumentation system(

Characteristic 1eatures o1 gel electrophoresis are,

a. ". c. d. e( f(

The molecular 6eight of the 2N <

the migration

g.

h. i. 0. ;(

rate is in3ersely proportional to the mol( wt( garose concentration< the migration rate is in"ersely proportional to the agarose concentration* Conformation of the 2N < linear form tra"els slo6est an# the supercoile# form tra"els fastest* pplie# "oltage< typical "alue is & ? per cm2* The heat generate# #uring electrophoresis is #issipate# !y the !uffer* 2N !eing polynaionic at neutral p/8 it migrates to6ar#s ano#e* The loa#ing #ye for 2N contains glycerol %6hich gi"es #ensity to help the samples sin; to the !ottom of the 6ell( an# mar;er #yes li;e ,ylene Cyanol an# !romophenol !lue* BPB mo"es on par 6ith $''--''!p an# @ylene cyanol 6ith 2-$;! 2N The 2N is "isuali:e# !y a##ing .tBr8 a fluorescent molecule 6hich intercalates 6ith the 2N !ases* To '*1+ agarose gel a## .tBr to a final concentration of '*&DgHml( A? ra#iation at 2&-nm is a!sor!e# !y the 2N an# transmitte# to the !oun# #ye* The energy is re-emitte# at &3'nm in a re#-orange region of the spectrum* .tBr is a po6erful mutagen* The #ye is usually incorporate# into the gel or con"ersely the gel is staine# after running !y soa;ing in a solution of .tBr* The usual sensiti"ity of #etection is '*'1Dg o1 6+2( The gel 6ill !e run along 6ith a molecular 6eight mar;er8 a 6i#e range of 6hich is commercially a"aila!le*

Protocol
a. Ta;e 1+ %6>"( agarose in Tris-acetate .2T !uffer %1, T .( an# heat to #issol"e the agarose* ". Cool the solution to -&-&'EC an# a## .tBr %'*& Bg>ml(* Mi@ 6ell* c( Before pouring the solution into gel casting tray 6ash the tray an# com! 6ith 248 an# place the tray on a le"ele# surface*

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#( Choose appropriate com! an# fi@ into position* e. Pour the gel into the apparatus an# allo6 it to cool an# set* f. fter the gel has set firmly8 pour little amount of !uffer an# remo"e the com! gently* Ta;e care not to #rag the com! an# !rea; the gel* g( Immerse the gel slo6ly into the gel tan;* ## sufficient amount of 1, T . !uffer* Connect the electro#e an# chec; the current* h( Loa# the samples into 6ells carefully* i( l6ays loa# an ali7uot of stan#ar# molecular 6eight mar;er along 6ith the samples* It 6ill help in assessing the si:e of the 2N fragment !y comparing 6ith the electrophoretic mo!ility*

nticipate# 9esults
2epen#ing upon its conformation an# si:e8 the 2N fragments 6ill mo"e as #iscrete !an#s* The .tBr intercalate# 6ith the 2N fragments 6ill ma;e it emit pin; fluorescence un#er A? light*

7 - 9uanti1ication o1 6+2
*ntro uction
Reliable measurement o1 6+2 concentration is important 1or many applications in >olecular Biology inclu ing complete igestion o1 6+2 by Restriction 8n=yme an ampli1ication o1 target 6+2 by PCR( 6+2 Kuanti1ication is generally carrie out by spectrophotometric measurements or by agarose gel analysis

Principle
Bhe purines an pyrimi ines in nucleic aci show absorption ma@ima aroun "#$nm & 2BP, "-7N CBP, "/"N GBP, "-4N BBP, "./) o1 the 6+2 samples in pure without any contamination o1 protein or organic sol3ents( So absorption at "#$nm can be taken to

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correctly assess the Kuantity( *1 the sample amount is less it can be 3isually estimate by gel electrophoresis(

Protocol
a. Bake 4 ml o1 B8 in two cu3ettes &one 1or sample an the other 1or re1erence) an calibrate the absorbance to =ero at "#$nm 5 ":$nm both, a1ter placing the cu3ettes in the slots( ". Replace -Dl o1 B8 with the same amount o1 total 6+2 1rom the sample cu3ette( Co3er it with para1ilm 5 mi@ properly by gently in3erting the cu3ette( c. Aipe the outer walls o1 the cu3ette with tissue paper an replace the cu3ette in its slot( Recor the absorbance at "#$nm an ":$nm( d. <ollow the same proce ure 1or the other samples( e) ;ne ;6 at "#$nm correspon s to -$DgHml o1 s6+2, 44DgHml o1 ss6+2, .$Dg o1 R+2 an "$ - 4$Dg o1 nucleoti es( 1) Let the absorbance at "#$nm be G 2 Since %($ absorbance at "#$nm correspon s to G -$($DgHml Cence 6+2 concentration G -$ 2 ilution 1actor &DgHml) g. Ju ge the Kuality o1 6+2 1rom the ratio o1 the ;6 3alues at "#$nm an ":$nm( Pure 6+2 has 2-(,H2-*, ratio o1 %(: an pure R+2 o1 aroun "($( Protein with max o1 ":$nm has an 2-(,H2-*, ratio o1 less than %($( Cence, a ratio o1 less than %(: suggests protein contamination an R+2 contamination i1 it is eKual or greater than "($

%$ - Restriction igestion o1 phage 6+2

*ntro uction
8n onucleases are en=ymes that pro uce internal cuts, calle clea3age, in 6+2 molecules( >any en onucleases clea3e 6+2 molecules at ran om sites, which ha3e speci1ic base seKuence, such en onucleases are known as restriction en onucleses an the sites recogni=e by them are calle recognition sites( Bhe recognition seKuences are i11erent an speci1ic 1or i11erent restriction en onucleases or restriction en=ymes(

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Reaction >i@ture
a. ". c. d. Lam a 6+2 Cin *** %$J reaction bu11er +uclease 1ree water <inal 3olume , , %$Dl "Dl , "Dl , #Dl , "$Dl

Protocol
a. Place the 3ial 1or reaction mi@ture on ice an a "Dl Cin *** en=yme ". Bhaw the 3ials containing 6+2 &substrate) an %$J assay bu11er( c. 2 %$Dl o1 6+2 in the 3ial( d. 2 "Dl o1 %$J reaction bu11er an #Dl o1 nuclease 1ree water in 3ial( >i@ these tubes by 3orte@ing( e. *ncubate the 3ial at 4/EC 1or % hr( 1) Prepare %F 2garose gel 1or electrophoresis( g. 21ter completing one hr o1 incubation an appropriate amount o1 gel loa ing bu11er in igeste sample an loa in 2garose gel( h) 2lso loa un igeste substrate 6+2 an marker in gel 1or comparison( i) 8lectrophorese the samples at -$-%$$? 1or %-%(- hrs( I) ?isuali=e the 6+2 ban s by '? Bransilluminator(

%% - Ligation o1 6+2 1ragments

*ntro uction
Construction o1 any recombinant molecule o1 6+2 is epen ent on ligation o1 -O phosphate an 4O hy ro@yl terminus( Bhe ligase en=yme catalyses the 1ormation o1 phospho iester bon s between -O phosphate an 4O hy ro@yl terminus o1 s6+2( Bhe B.

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6+2 ligase has the uniKue ability to Ioin sticky an 6+2 1ragments(

blunt en s o1

Reaction >i@ture
a. ". c. d. Lamb a 6+2 igest B. 6+2 ligase %$J reaction bu11er +uclease 1ree water <inal 3olume , , %$Dl "Dl , "Dl , #Dl , "$Dl

Protocol
a) Place the 3ial 1or reaction mi@ture on ice( ". Bake igeste lamb a 6+2 or igest it as earlier e@periment an a %$Dl o1 it in 3ial( c) Bhaw the 3ials containing B. 6+2 ligase an assay bu11er( d. 2 "Dl o1 B. 6+2 ligase an "Dl o1 assay bu11er in 3ial( e. 2 #Dl o1 nuclease 1ree water in mi@ture an mi@ it by 3orte@ing( f. *ncubate the reaction mi@ at 4/EC 1or " hrs in a pre set water bath 1or ligation( g) Prepare %F 2garose gel 1or electrophoresis( h. 21ter completing " hrs o1 incubation an appropriate amount o1 gel loa ing bu11er in ligate sample an loa in 2garose gel( i) 2lso loa igeste substrate 6+2 marker in gel 1or comparison( I) 8lectrophorese the samples at -$-%$$? 1or %-%(- hrs( k) ?isuali=e the 6+2 ban s by '? Bransilluminator(

%" - Polymerase Chain Reaction

*ntro uction
PCR &in3ente by Kary >ullis, %7:$) is an in vitro metho by which speci1ic 6+2 segments that lies between two regions o1 known seKuences or primer bin ing sites are selecti3ely ampli1ie

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se3eral 1ol s in the presence o1 thermostable 6+2 polymerase( PCR is typically carrie out using two oligonucleoti e primers &one 1orwar an one re3erse) that 1lank the 6+2 1ragments to be ampli1ie ( Bhese primers hybri i=e with opposite stran s o1 the target seKuence an are oriente so that 6+2 synthesis by the polymerase procee s across the region between the primers( *n general the ampli1ication in3ol3es three basic steps, which are , &i) enaturation o1 template 6+2 in the presence o1 large molar e@cess o1 each o1 +BPs an primer( &ii) annealing o1 the primer to the complimentary region on the template 6+2 an 1inally &iii) e@tension o1 the anneale primers with thermostable 6+2 Polymerase( 2s the e@tension pro ucts are complimentary to an capable o1 bin ing primers, successi3e cycles o1 ampli1ication ouble the amount o1 target 6+2 synthesi=e in the pre3ious cycles thus resulting in rapi e@ponential accumulation o1 the speci1ic target 6+2( Bhe termini o1 this e@ponential reaction & s6+2 segment) are e1ine by the -P termini o1 the primer an length by the istance between the primer bin ing sites( Cowe3er, pro ucts o1 1irst cycle are heterogeneously si=e , an the length may e@cee istance between the primer bin ing sites( *n secon cycle 6+2s o1 e1ine length are generate which are e@ponentially accumulate in late cycles o1 ampli1ication an constitute the ominant pro uct o1 PCR( Because longer molecules accumulate at a linear rate, they o not signi1icantly contribute to the 1inal mass o1 pro ucts( Since primer e@tension an annealing is carrie out at ele3ate temperatures, the pro ucts become speci1ic, the si=e an yiel is also impro3e ( 2lthough e@tremely e11icient un er normal reaction con itions the amount o1 BaK 6+2 Polymerase becomes limiting 1actor a1ter 4$ cycles o1 ampli1ication

Reagents
&i) &ii) &iii) 2iv. &3) &3i) 2vii. Bemplate 6+2 Primers +BPs >gCl" %$J PCR bu11er BaK 6+2 Polymerase >ineral oil &optional)

Protocol
6+2 samples &%" ng) are ampli1ie in %-Dl reaction mi@tures containing %$J PCR bu11er &%$ m> Bris &pC G 7($), -$ m> KCl, %(/ m> >gCl", $(% F Gelatin), $(%"- m> o1 each o1 the +BPs, 7 ng

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primer an * unit o1 BaK 6+2 polymerase( 2 cocktail mi@ is prepare 1or reKuire number o1 reactions plus one reaction to compensate the pipetting loss( Bhis e@tra reaction mi@ is use as blank &without 6+2) so as to check arti1acts, i1 any( a. <or n no o1 reactions prepare a cocktail mi@ as 1ollows&%-Dl), Bu11er %(-Dl @ &nQ%)N +BPs %(-Dl @ &nQ%)N >gCl" $(%"Dl @ &nQ%)N primer %(:Dl @ &nQ%)N BaK 6+2 polymerase &4 unitsHDl) $(4Dl @ &nQ%)N sterile 6A :(-:Dl @ &nQ%)( ". 2rrange $(- ml thin walle 1lat cap PCR tubes in rack an label them 1or each genomic 6+2( c. 2 %("Dl &%" ng) o1 each genomic 6+2 to the correspon ing PCR tubes arrange in the rack( d. 2 %4(:Dl o1 the cocktail mi@ to each o1 the PCR tube containing genomic 6+2( e) ;ptional step i1 the thermal cycler is not eKuippe with heate li , o3erlay a rop o1 mineral oil to the reaction mi@ to pre3ent e3aporation o1 reaction mi@ at higher temperature uring PCR cycles( f. Close the caps an gi3e a momentary spin( g. Place the tubes in the thermal cycler an set it to carry out the 1ollowing temperature pro1ile, initial enaturing at 7-RC 1or - min, 1ollowe by 4$ cycles o1 enaturation at 7-RC 1or % min, annealing at --RC 1or % min 5 primer e@tension at /"RC 1or " min an 1inally primer e@tension at /"RC 1or / min 5 storing at .RC( h) Separate PCR pro ucts on %(-F 2garose gel( i) ?isuali=e an recor the ata with the help o1 Gel 6ocumentation system(

%4 - 8lution o1 6+2 1rom 2garose Gels

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Genetech Laboratories

*ntro uction
Ahen a speci1ic 6+2 o1 the same si=e is nee e , then there is nee to isolate or elute the 6+2 1rom gel 1or 1urther stu y( <or e@ample a1ter PCR ampli1ication o1 6+2 you will 1in se3eral ban s in R2P6 or speci1ic ban s along with some non speci1ic ban s with gene speci1ic primers( *n such cases you may reKuire to elute the esire 6+2 1ragments(

Protocol
a) 8@cise the 6+2 1ragment 1rom the 2garose gel with a clear, sharp scalpel( Aeigh the gel slice an trans1er it to a %(-ml centri1uge tube( ". 2 4$$Dl o1 +8 bin ing bu11er to the centri1uge tube containing %$$mg gel slice( *ncubate at -$-#$RC 1or 4-- min an in3ert the tube occasionally until the 2garose gel is completely issol3e ( c) Brans1er the abo3e mi@ture to a miniprep spin column with a "ml collection tube( Let it stan 1or - min( centri1uge at %4$$$rpm 1or %$-"$ sec an iscar the 1low through( d. 2 -$$Dl o1 :$F isopropanol &or ethanol) to the spin column( Centri1uge at %4$$$rpm 1or 4$ sec an iscar the 1low through( e) Repeat the washing proce ure in step )( f. Centri1uge at %4$$$rpm 1or an a itional % minute to remo3e the resi ual isopropanol( g. Place the spin column into a new %(-ml micro1uge tube( Let the tube li open 1or "-4 min to 3olatili=e isopropanol completely( h. 2 -$Dl B8 bu11er or ultrapure water into the center part o1 the sima@ membrane in a spin column( *ncubate at RB 1or 4-min( Centri1uge at %4$$$rpm 1or % min to elute 6+2( i. 6etermine the Kuality o1 6+2 1ragments on %F 2garose gel staine with 8tBr( Store the puri1ie 6+2 at .RC 1or imme iate use or at -"$RC 1or 1uture use(

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%. - S6S-P2G8
*ntro uction
So ium 6o ecyl Sulphate-Polyacrylami e Gel 8lectrophoresis &S6S-P2G8) is the most wi ely use metho 1or Kuantitati3e analysis o1 protein samples( Bhe separation o1 proteins is base on the si=e o1 the protein, hence this metho can be use to etermine the molecular weight o1 a gi3en protein sample( S6S use is an anionic etergent( Bhe samples to be run are boile 1or - min in a sample bu11er containing / mercaptoethanol, S6S an P>S<( / mercaptoethanol is use to re uce any isulphi e bri ges that are hol ing tertiary structure together( S6S enatures the protein an opens the protein into a ro shape structure with a series o1 negati3ely charge S6S molecules along the polypepti e chain(one S6S molecule bin s 1or e3ery two amino aci resi ue( Sample bu11er also contains an ionisable tracking ye, usually BPB, that allows the electrophoretic run to be monitore an sucrose or glycerol so that the sample can settle own in the well when loa e

Purpose o1 stacking gel

Stacking gel is use to concentrate the protein sample into sharp ban be1ore it enters the resol3ing gel( Bhis is achie3e by utili=ing the i11erences in ionic strength an pC between the electrphoretic bu11er an the stacking gel, which in3ol3es a phenomenon known as istachophoresis( Stacking gel has larger pore si=e, which allows the protein samples to concentrate an mo3e 1reely un er the e11ect o1 electric 1iel ( Ban sharpening is attaine by the i11erence in the electrophoretic mobility o1 glycinate ions, protein-S6S comple@ an chlori e ions in the loa ing bu11er( <or ha3ing a stea y electric circuit all the species ha3e to migrate at the same spee un er the in1luence o1 the applie 1iel ( <iel strength is in3ersely proportional to con ucti3ity that is proportional to concentration( Cl S Protein-S6S S Glycinate 6ue to lower concentration o1 protein-S6S comple@ they concentrate in a 3ery tight ban between glycinate an chlori e ion boun aries( ;nce glycinate ions reach the resol3ing gel ue to higher pC en3ironment they get easily ioni=e an their mobility increases(

Purpose o1 resol3ing gel

;nce the protein-S6S comple@ enters the resol3ing gel, owing to molecular sie3ing property o1 the gel, separation o1 the proteins in the sample is obser3e ( Smaller proteins mo3e more easily an hence mo3e 1arther when compare to larger proteins( BPB being small molecule mo3es 1arther an 1orms electrophoretic 1ront(

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Sample preparation
Samples 1or S6S-P2G8 are prepare by a ing %J sample bu11er to the e@tracte protein samples( Bhe samples are centri1uge an then the supernatant is trans1erre into an eppen or1 tube( Bhe samples are store at -:$EC( Just be1ore running, the samples are boile 1or - min an place on ice till loa ing(

Protocol
a) b) c. ) e. Clean the glass plates be1ore setting the gel moul ( 'se silicone as grease an apply on all spacers to be use ( 2pply spacers on their respecti3e position( Seal the glass plates 1rom three si es( <irst prepare resol3ing gel an pour in the gap between the two plates( f. 21ter the resol3ing gel polymeri=es, prepare stacking gel an pour it so that it layers on top o1 resol3ing gel( g. Place the comb imme iately a1ter pouring the stacking gel( h. Lea3e the setup 1or hal1 an hour 1or the stacking gel to polymeri=e an remo3e the comb upon polymeri=ation o1 the gel( i) 2 Iust the S6S-P2G8 apparatus in such a way that it is parallel to the groun ( 0. Remo3e the lower spacer, 1i@ the gel to the apparatus using silicone grease, an tighten the screws( k) 2 %J running bu11er to the apparatus( l) Run the gel without loa ing sample 1or - -%$ min( m) Prepare an loa the samples an run at appropriate 3oltage till the ye runs out( n) Remo3e the gel moul , trans1er it into a tray with staining solution, an lea3e o3ernight with intermittent rocking( o) Brans1er the gel into the estaining solution till the ban s appear properly(

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