instruction manual - uni-koeln.de · la jolla, ca 92037 telephone (858) 535-5400 order toll free...

FairPlay™ Microarray Labeling Kit

INSTRUCTION MANUAL

`~í~äçÖ=@OROMMO=ENM=êÉ~ÅíáçåëF=~åÇ=@OROMMP=EPM=êÉ~ÅíáçåëF

oÉîáëáçå=@MQOMMR

For In Vitro Use Only

*252002-12__042005/*

LIMITED PRODUCT WARRANTY

qÜáë=ï~êê~åíó= äáãáíë= çìê= äá~Äáäáíó= íç= êÉéä~ÅÉãÉåí= çÑ= íÜáë= éêçÇìÅíK= kç= çíÜÉê=ï~êê~åíáÉë= çÑ= ~åóâáåÇI=ÉñéêÉëë=çê=áãéäáÉÇI=áåÅäìÇáåÖ=ïáíÜçìí=äáãáí~íáçåI=áãéäáÉÇ=ï~êê~åíáÉë=çÑ=ãÉêÅÜ~åí~Äáäáíó=çêÑáíåÉëë=Ñçê=~=é~êíáÅìä~ê=éìêéçëÉI=~êÉ=éêçîáÇÉÇ=Äó=píê~í~ÖÉåÉK=píê~í~ÖÉåÉ=ëÜ~ää=Ü~îÉ=åç=äá~Äáäáíó=Ñçê~åó=ÇáêÉÅíI= áåÇáêÉÅíI=ÅçåëÉèìÉåíá~äI=çê= áåÅáÇÉåí~ä=Ç~ã~ÖÉë=~êáëáåÖ=çìí=çÑ= íÜÉ=ìëÉI= íÜÉ= êÉëìäíë=çÑìëÉI=çê=íÜÉ=áå~Äáäáíó=íç=ìëÉ=íÜáë=éêçÇìÅíK

ORDERING INFORMATION AND TECHNICAL SERVICES

United States and CanadaStratagene

11011 North Torrey Pines Road

La Jolla, CA 92037

Telephone (858) 535-5400

Order Toll Free (800) 424-5444

Technical Services (800) 894-1304

Internet [email protected]

World Wide Web www.stratagene.com

Stratagene European Contacts

Location Telephone Fax Technical Services

Austria 0800 312 526 0800 312 527 017 956 7036

00800 7000 7000 00800 7001 7001 00800 7400 7400Belgium

0800 96078 0800 96024 027 13 12 11

France 00800 7000 7000 00800 7001 7001 00800 7400 7400

0800 100391 0800 881323

Germany 00800 7000 7000 00800 7001 7001 00800 7400 7400

06221 400 634 06221 400 639 06221 400 637

0130 840 911 0130 762 088 069 9509 6197

Netherlands 00800 7000 7000 00800 7001 7001 00800 7400 7400

0800 023 0446 0800 023 0447 0800 023 0448

Switzerland 00800 7000 7000 00800 7001 7001 00800 7400 7400

061 6930 540 061 6930 544 01 800 9045

0800 830 250 0800 825 225

United Kingdom 00800 7000 7000 00800 7001 7001 00800 7400 7400

01223 420 955 01223 420 234 0171 365 1056

0800 585 370 0800 783 0889

All Other CountriesPlease contact your local distributor. A complete list of distributors is available at www.stratagene.com.


CONTENTS

j~íÉêá~äë=mêçîáÇÉÇ KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==N

píçê~ÖÉ=`çåÇáíáçåë KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==N

^ÇÇáíáçå~ä=j~íÉêá~äë=oÉèìáêÉÇ KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==N

^ÇÇáíáçå~ä=j~íÉêá~äë=oÉèìáêÉÇ=Ñçê=eóÄêáÇáò~íáçå=íç=jáÅêç~êê~ó KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==O

kçíáÅÉ=íç=mìêÅÜ~ëÉê KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==O

fåíêçÇìÅíáçå KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==P

mêçíçÅçä=dìáÇÉäáåÉë KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==Q

mêçíçÅçäKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==S

Åak^=dÉåÉê~íáçå KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==S

Åak^=mìêáÑáÅ~íáçå KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==T

kepJbëíÉê=`çåí~áåáåÖ=aóÉ=`çìéäáåÖ=oÉ~Åíáçå= Ecçê=rëÉ=ïáíÜ=^ãÉêëÜ~ã=mÜ~êã~Åá~

_áçíÉÅÜ=aóÉëFKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==U

aóÉJ`çìéäÉÇ=Åak^=mìêáÑáÅ~íáçåKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK ==V

^å~äóëáë=çÑ=cäìçêÉëÅÉåÅÉJä~ÄÉäÉÇ=Åak^=EoÉÅçããÉåÇÉÇF KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKNO

eóÄêáÇáò~íáçå KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKNP

t~ëÜáåÖ KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKNQ

eóÄêáÇáò~íáçå=aÉíÉÅíáçåKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKNQ

qêçìÄäÉëÜççíáåÖKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKNR

mêÉé~ê~íáçå=çÑ=oÉ~ÖÉåíëKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKNS

^ééÉåÇáñ=fKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKNT

oÉÑÉêÉåÅÉë KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKNT

båÇåçíÉë KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKNT

FairPlay™ Microarray Labeling Kit 1


MATERIALS PROVIDED

Quantity

Material providedCatalog #252002

10 reactions

Catalog #252003

30 reactions

10× StrataScript™ reaction buffer 50 µl 150 µl

20× dNTP mix with amino allyl dUTP 10 µl 30 µl

Oligo d(T)12–18

primer (500 ng/µl) 20 µl 60 µl

0.1 M DTT 30 µl 90 µl

RNase block (40 U/µl) 5 µl 15 µl

StrataScript™ reverse transcriptase (50 U/µL) 20 µl 60 µl

DEPC water 500 µl 1.5 ml

20 mg/ml Glycogen 10 µl 30 µl

2× Coupling Buffer 100 µl 300 µl

DMS0 (high purity) 500 µl 1.5 ml

DNA-binding solution 2.5 ml 5.0 ml

Microspin cupsa and 2-ml receptacle tubes 10 30b The capacity of the microspin cup is ~0.8 ml.

STORAGE CONDITIONS

ak^J_áåÇáåÖ=pçäìíáçåI=jáÅêçëéáå=`ìéë=~åÇ=oÉÅÉéí~ÅäÉ=qìÄÉëW=oççã=qÉãéÉê~íìêÉ

^ää=líÜÉê=`çãéçåÉåíëW=ÓOMø`

ADDITIONAL MATERIALS REQUIRED

^ãÉêëÜ~ã=mÜ~êã~Åá~=_áçíÉÅÜ=kepJbëíÉê=aóÉë

cäìçêçiáåâ»=`v»=P=ãçåçÑìåÅíáçå~ä=ÇóÉ=RJé~Åâ=E`~í~äçÖ=@mÔPMMNFcäìçêçiáåâ�»=`v»=PKR=ãçåçÑìåÅíáçå~ä=ÇóÉ=RJé~Åâ=E`~í~äçÖ=@mÔPRMNFcäìçêçiáåâ»=`v»=R=ãçåçÑìåÅíáçå~ä=ÇóÉ=RJé~Åâ=E`~í~äçÖ=@mÔRMMNFcäìçêçiáåâ»=`v»=RKR=ãçåçÑìåÅíáçå~ä=ÇóÉ=RJé~Åâ=E`~í~äçÖ=@mÔRRMNF

jçäÉÅìä~ê=mêçÄÉë=pqmJbëíÉê=aóÉë

ôbp»=^äÉñ~=cäìçê∆=PRM=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSTRFôbp»=lêÉÖçå=dêÉÉå∆=QUU=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSTQFôbp»=^äÉñ~=cäìçê∆=QUU=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSSRFôbp»=^äÉñ~=cäìçê∆=RPO=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSSSFôbp»=^äÉñ~=cäìçê∆=RQS=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSSTFôbp»=^äÉñ~=cäìçê∆=RRR=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSTTFôbp»=^äÉñ~=cäìçê∆=RSU=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSSUFôbp»=^äÉñ~=cäìçê∆=RVQ=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSSVFôbp»=^äÉñ~=cäìçê∆=SQT=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSTSF

oÉîáëáçå=@MQOMMR `çéóêáÖÜí=«=OMMO=Äó=píê~í~ÖÉåÉK

2 FairPlay™ Microarray Labeling Kit

ADDITIONAL MATERIALS REQUIRED (CONTINUED)N=j=k~leN=j=e`äNMMB=bíÜ~åçäI=ãçäÉÅìä~ê=ÄáçäçÖó=Öê~ÇÉI=ak~ëÉLok~ëÉ=ÑêÉÉVRB=bíÜ~åçäI=ãçäÉÅìä~ê=ÄáçäçÖó=Öê~ÇÉI=ak~ëÉLok~ëÉ=ÑêÉÉTRB=bíÜ~åçäI=ãçäÉÅìä~ê=ÄáçäçÖó=Öê~ÇÉI=ak~ëÉLok~ëÉ=ÑêÉÉ=EëÉÉ=�jáÅêçëéáå=`ìé=t~ëÜ=_ìÑÑÉêÒF¬

TMB=bíÜ~åçäI=ãçäÉÅìä~ê=ÄáçäçÖó=Öê~ÇÉI=ak~ëÉLok~ëÉ=ÑêÉÉok~ëÉLak~ëÉJÑêÉÉ=eOlI=xoÉëÉ~êÅÜ=dÉåÉíáÅëI=fåÅKI=`~í~äçÖ=@TRMMOPzqêáëJ~ÅÉí~íÉ=~Ö~êçëÉ=ÖÉä=EëÉÉ=^ééÉåÇáñFjáåáã~ä=ÇóÉ=äç~ÇáåÖ=ÄìÑÑÉê¬

ak^=ëáòÉ=ëí~åÇ~êÇI=ìëÉÑìä=ê~åÖÉ=QMMÓNMMM=ÄébíÜáÇáìã=ÄêçãáÇÉrs=íê~åëáääìãáå~íçêNM=ãj=qêáë=Ä~ëÉI=~Çàìëí=ée=íç=UKR=ïáíÜ=e`ä=EëÉÉ=�jáÅêçëéáå=`ìé=bäìíáçå=_ìÑÑÉêÒF¬

ADDITIONAL MATERIALS REQUIRED FOR HYBRIDIZATION TO

MICROARRAY

OM�=pp`=ÄìÑÑÉêI=ÜáÖÜJèì~äáíóI=ok~ëÉLak~ëÉJÑêÉÉ¬

NMB=EïLîF=pap=ëçäìíáçåI=ÜáÖÜJèì~äáíóI=ok~ëÉLak~ëÉJÑêÉÉ¬

ok~ëÉLak~ëÉJÑêÉÉ=eOlI=xoÉëÉ~êÅÜ=dÉåÉíáÅëI=fåÅKI=`~í~äçÖ=@TRMMOPzmçäó=ÇE^FQMÓSMI=U=”ÖL”ä=x^ãÉêëÜ~ã=mÜ~êã~Åá~=_áçíÉÅÜI=`~í~äçÖ=@OTJTVUUJMNzvÉ~ëí=íok^I=Q”ÖL”ä=xiáÑÉ=qÉÅÜåçäçÖáÉë=fåÅKI=`~í~äçÖ=@NRQMNJMNNzjáÅêç~êê~ó=ÜóÄêáÇáò~íáçå=ÅÜ~ãÄÉêëjáÅêç~êê~ó=ëÅ~ååÉêeìã~å= `lqJN= ak^∆I= NM= ”ÖL”ä¬= xfåîáíêçÖÉå= `çêéçê~íáçåI= E`~í~äçÖ= @NROTVJMNNFz= çê

jçìëÉ=`lqJN=ak^∆=xfåîáíêçÖÉå=`çêéçê~íáçåI=E`~í~äçÖ=@NUQQMJMNSFz

NOTICE TO PURCHASER

pçãÉ=ìëÉë=çÑ=íÜÉ=c~áêmä~ó»=ãáÅêç~êê~ó=ä~ÄÉäáåÖ=âáí=ã~ó=êÉèìáêÉ=äáÅÉåëÉë=Ñêçã=íÜáêÇ=é~êíáÉë=áå=ÅÉêí~áåÅçìåíêáÉëK

¬ pÉÉ=mêÉé~ê~íáçå=çÑ=jÉÇá~=~åÇ=oÉ~ÖÉåíëK


INTRODUCTION

^å=áåÅêÉ~ëáåÖ=íêÉåÇ=áå= áÇÉåíáÑóáåÖ=ÇáÑÑÉêÉåíá~ääó=ÉñéêÉëëÉÇ=ÖÉåÉë= áë= íÜÉ=ìëÉçÑ= åìÅäÉáÅ= ~ÅáÇ= ãáÅêç~êê~óë= íÜ~í= Åçåí~áå= ÜìåÇêÉÇë= çê= íÜçìë~åÇë= çÑ= éêçÄÉÖÉåÉëK

1= få= íÜÉëÉ= ÉñéÉêáãÉåíëI= íÉëí= ~åÇ= Åçåíêçä= ãok^ë= ~êÉ= ÅçåîÉêíÉÇ= Äó

êÉîÉêëÉ= íê~åëÅêáéíáçå= áåíç= Åak^ë= ïáíÜ= ä~ÄÉäÉÇ= åìÅäÉçíáÇÉë= Eí~êÖÉí= ak^FKqÜÉ=ä~ÄÉäÉÇ=íÉëí=~åÇ=Åçåíêçä=Åak^ë=~êÉ=íÜÉå=ÜóÄêáÇáòÉÇ=íç=éêçÄÉ=ÖÉåÉë=çåëÉé~ê~íÉ=ãáÅêç~êê~óëI=~åÇ=~ÑíÉê=ìåÜóÄêáÇáòÉÇ=Åak^=áë=êÉãçîÉÇI=ëáÖå~ä=ÑêçãíÜÉ=ÜóÄêáÇáòÉÇ=Åak^=áë=ÇÉíÉÅíÉÇK=aáÑÑÉêÉåÅÉë=áå=ÜóÄêáÇáò~íáçå=ëáÖå~äë=çå=íÜÉãáÅêç~êê~ó= ÅçêêÉä~íÉ=ïáíÜ= ÇáÑÑÉêÉåÅÉë= áå= ~ÄìåÇ~åÅÉ= çÑ= íÜÉ=ãok^= ìëÉÇ= íçéêÉé~êÉ=íÜÉ=ä~ÄÉäÉÇ=Åak^K

s~êáçìë= ä~ÄÉäáåÖ= éêçíçÅçäë= Ü~îÉ= ÉãÉêÖÉÇ= íç= ÅçåîÉêí= íÉëí= ~åÇ= êÉÑÉêÉåÅÉãok^= áåíç= ä~ÄÉäÉÇ= Åak^= áå= ~= êÉîÉêëÉ= íê~åëÅêáéíáçå= êÉ~ÅíáçåK=låÉ= çÑ= íÜÉãçêÉ=éçéìä~ê=ãÉíÜçÇë=áë=íÜÉ=ÇáêÉÅí=áåÅçêéçê~íáçå=çÑ=ä~ÄÉäÉÇ=åìÅäÉçíáÇÉë=áåíçíÜÉ= Åak^= ÇìêáåÖ= íÜÉ= êÉîÉêëÉ= íê~åëÅêáéíáçå= EoqF= êÉ~ÅíáçåK= ^å= áåÜÉêÉåíéêçÄäÉã= ~ëëçÅá~íÉÇ= ïáíÜ= íÜáë= ãÉíÜçÇ= áë= íÜÉ= Äá~ëÉÇ= áåÅçêéçê~íáçå= çÑ= íÜÉÇáÑÑÉêÉåí=`vJÇóÉëI=ïÜáÅÜ=Å~å= êÉëìäí= áå=ìåÉîÉå=ÇáëíêáÄìíáçå=çÑ= ÑäìçêÉëÅÉåÅÉ~åÇLçê=çîÉê~ää= äçïJäÉîÉä= ÑäìçêÉëÅÉåÅÉ= áå= íÜÉ= êÉëìäíáåÖ= Åak^K=qÜáë=ã~ó=ÄÉÇìÉ= íç= ÉáíÜÉê= íÜÉ= áå~Äáäáíó= çÑ= íÜÉ= oq= íç= ÉÑÑáÅáÉåíäó= áåÅçêéçê~íÉ= íÜÉ= Äìäâó`vJä~ÄÉäÉÇ= Çrqm= çê= íÜÉ= áåëçäìÄáäáíó= çÑ= íÜÉ= `vJä~ÄÉäÉÇ= Çrqm= ìåÇÉê= oqêÉ~Åíáçå=ÅçåÇáíáçåëK

píê~í~ÖÉåÉ=çÑÑÉêë= íÜÉ=c~áêmä~ó»=ãáÅêç~êê~ó= ä~ÄÉäáåÖ= âáí= Ñçê=ìëÉ=ïáíÜ=ÖÉåÉãáÅêç~êê~óë=íç=áåÅêÉ~ëÉ=íÜÉ=êÉäá~Äáäáíó=~åÇ=êÉéêçÇìÅáÄáäáíó=çÑ=Åak^=ä~ÄÉäáåÖãÉíÜçÇëK= qÜáë= âáí= ~ÇÇêÉëëÉë= íÜÉ= éêçÄäÉãë= ~ëëçÅá~íÉÇ= ïáíÜ= ÇáêÉÅíáåÅçêéçê~íáçå=Äó=ÇáîáÇáåÖ=íÜÉ=ä~ÄÉäáåÖ=éêçÅÉÇìêÉ=áåíç=íïç=é~êíëW=NF=éêÉé~êáåÖ~ãáåç=~ääóä=ãçÇáÑáÉÇ=Åak^=~åÇ=OF=ÅÜÉãáÅ~ääó=ÅçìéäáåÖ=íÜÉ=`vJÇóÉ=íç=íÜÉãçÇáÑáÉÇ=Åak^K=qÜÉ=âáí=Åçåí~áåë=ÅçãéçåÉåíë=åÉÅÉëë~êó=íç=ÅçåîÉêí=ok^=íç~ãáåç= ~ääóä= ãçÇáÑáÉÇ= ÅakÎ= êÉ~Çó= íç= ÄÉ= ÅçìéäÉÇ= íç= ~åó= ÑäìçêÉëÅÉåí= ÇóÉÅçåí~áåáåÖ= ~= kepJ= çê= pqmJÉëíÉê= äÉ~îáåÖ= ÖêçìéK= qÜÉëÉ= áåÅäìÇÉ= íÜÉ

ÑäìçêÉëÅÉåí= ÇóÉë= áå= íÜÉ= ôbp= ^äÉñ~= cäìçê= ak^= i~ÄÉäáåÖ= háíëEjçäÉÅìä~ê= mêçÄÉëF= ~åÇ= ãçåçJêÉ~ÅíáîÉ= ÇóÉ= é~Åâë= E^ãÉêëÜ~ã= mÜ~êã~Åá~_áçíÉÅÜFK

qç= ÖÉåÉê~íÉ= ÑäìçêÉëÅÉåÅÉJä~ÄÉäÉÇ= ÅakÎ= íçí~ä= ok^= áë= ~ååÉ~äÉÇ= íç= ~åçäáÖçåìÅäÉçíáÇÉ= ÇEqF= éêáãÉêI= ~åÇ= Åak^= áë= ëóåíÜÉëáòÉÇ= áå= ~= êÉîÉêëÉíê~åëÅêáéíáçå= êÉ~Åíáçå= ïáíÜ= píê~í~pÅêáéí»= êÉîÉêëÉ= íê~åëÅêáéí~ëÉEpíê~í~pÅêáéí=oqFI= ìåä~ÄÉäÉÇ= ÇkqmëI= ~åÇ= ~å= ~ãáåç= ~ääóä= ãçÇáÑáÉÇ= ÇrqmKqÜÉ=~ãáåç=~ääóä=ä~ÄÉäÉÇ=Åak^=áë=íêÉ~íÉÇ=ïáíÜ=k~le=íç=ÜóÇêçäóòÉ=íÜÉ=ok^ëíê~åÇK=qÜÉ=ä~ÄÉäÉÇ=Åak^=áë=íÜÉå=éìêáÑáÉÇ=íç=êÉãçîÉ=ìåáåÅçêéçê~íÉÇ=ÇkqmëIÜóÇêçäóòÉÇ=okÎ=~åÇ=~åó=qêáë=çê=çíÜÉê=ÅçãéçåÉåíë=çÑ=íÜÉ=ä~ÄÉäáåÖ=êÉ~ÅíáçåíÜ~í=ïçìäÇ= áåÜáÄáí= íÜÉ=ÅçìéäáåÖ= êÉ~ÅíáçåK=qÜÉ=~ãáåç=~ääóä= ä~ÄÉäÉÇ=Åak^=áëíÜÉå= ÅçìéäÉÇ= íç= ~= ÑäìçêÉëÅÉåí= ÇóÉ= Åçåí~áåáåÖ= ÉáíÜÉê= ~= kepJ= çê= pqmJÉëíÉêäÉ~îáåÖ= ÖêçìéK= qÜÉ= ÑäìçêÉëÅÉåí= ÇóÉJä~ÄÉäÉÇ= Åak^= áë= éìêáÑáÉÇ= EìëáåÖ= íÜÉak^JÄáåÇáåÖ=ëçäìíáçå=~åÇ=ãáÅêçëéáå=Åìéë=éêçîáÇÉÇF= íç=êÉãçîÉ=ìåÅçìéäÉÇÑäìçêÉëÅÉåí=ÇóÉ=éêáçê=íç=ÜóÄêáÇáò~íáçåK


qç=éêÉé~êÉ= Ñçê= íÜÉ=ÜóÄêáÇáò~íáçå=ëíÉéI= íÜÉ= ä~ÄÉäÉÇ= Åak^= áë= íÜÉå=ÅçãÄáåÉÇïáíÜ=ÄäçÅâáåÖ=ak^I=pp`=ÄìÑÑÉêI=~åÇ=papK=qÜÉ=ä~ÄÉäÉÇ=Åak^=áë=ÜóÄêáÇáòÉÇíç= íÜÉ=éêçÄÉ=ak^=çå=ãáÅêç~êê~óë=Äó= áåÅìÄ~íáåÖ=~í= SRø`=Ñçê=NQÓNU=ÜçìêëKqÜÉ=ìåÜóÄêáÇáòÉÇ= ä~ÄÉäÉÇ=Åak^=áë=êÉãçîÉÇ=áå= íïç=ï~ëÜáåÖ=ëíÉéë= ~åÇ= íÜÉÜóÄêáÇáòÉÇ=ä~ÄÉäÉÇ=Åak^=áë=îáëì~äáòÉÇ=ìëáåÖ=~=ãáÅêç~êê~ó=ëÅ~ååÉêK=péÉÅáÑáÅak^= ÜóÄêáÇáò~íáçå= ÄÉíïÉÉå= íÜÉ= ä~ÄÉäÉÇ= Åak^= ~åÇ= éêçÄÉ= ak^= çå= íÜÉãáÅêç~êê~ó= áë= éêçãçíÉÇ=ïÜáäÉ= ìåÇÉëáêÉÇ=ak^=ÜóÄêáÇáò~íáçå= áë= êÉÇìÅÉÇ=ÄóíÜÉ= ~ÇÇáíáçå= çÑ= ÄäçÅâáåÖ= ak^= EëÉÉ= mêçíçÅçä= dìáÇÉäáåÉë= Ñçê= ãçêÉáåÑçêã~íáçå=çå=ÄäçÅâáåÖ=ak^=ëçìêÅÉëFK

PROTOCOL GUIDELINES

♦ qÜÉ= èì~äáíó= ~åÇ= èì~åíáíó= çÑ= íÜÉ= ok^= ìëÉÇ= áë= ÅêáíáÅ~ä= Ñçê= ëìÅÅÉëëÑìäéêÉé~ê~íáçå= çÑ= ä~ÄÉäÉÇ= Åak^K= qÜÉ= éêÉëÉåÅÉ= çÑ= ÅÉääìä~ê= äáéáÇëIÅ~êÄçÜóÇê~íÉë= çê= éêçíÉáåë= ïáää= ëáÖåáÑáÅ~åíäó= áåÅêÉ~ëÉ= Ä~ÅâÖêçìåÇÑäìçêÉëÅÉåÅÉ=ÑçääçïáåÖ=ÜóÄêáÇáò~íáçåK=qÜÉ=la=OSMLOUM=ê~íáç=çÑ=íÜÉ=ok^ãìëí=ÄÉ=[NKUK=tÜÉå=ok^=áëçä~íÉÇ=Ñêçã=ã~ãã~äá~å=ëçìêÅÉë=áë=îáÉïÉÇçå=~=ÇÉå~íìêáåÖ=~Ö~êçëÉ=ÖÉäI=íÜÉ=êáÄçëçã~ä=Ä~åÇë=EOUp=~åÇ=NUpF=ëÜçìäÇ~ééÉ~ê=~ë=íïç=ÄêáÖÜí=Ä~åÇë=~í=~ééêçñáã~íÉäó=QKR=~åÇ=NKV=âáäçÄ~ëÉëK=qÜÉê~íáç=çÑ=áåíÉåëáíáÉë=çÑ=íÜÉ=OUp=~åÇ=NUp=Ä~åÇë=ëÜçìäÇ=ÄÉ=NKRJOKRWNK=içïÉêê~íáçë= ã~ó= áåÇáÅ~íÉ= íÜ~í= ok^= ÇÉÖê~Ç~íáçå= Ü~ë= çÅÅìêêÉÇ= ~åÇ= íÜ~í= íÜáëok^= ã~ó= åçí= ÄÉ= ëìáí~ÄäÉ= Ñçê= íÜÉ= éêÉé~ê~íáçå= çÑ= ä~ÄÉäÉÇ= Åak^K^ÇÇáíáçå~ä=Ä~åÇëI=áåÅäìÇáåÖ=äçï=ãçäÉÅìä~ê=ïÉáÖÜí=Ä~åÇë=ÅçêêÉëéçåÇáåÖíç= íÜÉ= Rp= êáÄçëçã~ä= ok^= ~åÇ= íok^= ã~ó= ~äëç= ÄÉ= îáëáÄäÉK= qÜÉÖì~åáÇáåáìã= áëçíÜáçÅó~å~íÉ=ãÉíÜçÇ=ìëÉÇ= áå= íÜÉ=píê~í~mêÉé∆= íçí~ä=ok^ãáåáéêÉé= âáí= áë= áÇÉ~ä= Ñçê= íÜÉ= áëçä~íáçå= çÑ= íçí~ä= ok^= Ñçê= ìëÉ= áå= íÜáë~ééäáÅ~íáçåK

♦ qÜÉ=éêÉëÉåÅÉ=çÑ=baq^=~åÇLçê=ÉíÜ~åçä=Å~å=áåÜáÄáí=êÉîÉêëÉ= íê~åëÅêáéí~ëÉ~Åíáîáíó=áå=íÜÉ=ä~ÄÉäáåÖ=êÉ~ÅíáçåK=tÜáäÉ=ïÉ=êÉÅçããÉåÇ=íÜ~í=íÜÉ=ok^=ÄÉÉíÜ~åçä= éêÉÅáéáí~íÉÇI= ~ää= ÉíÜ~åçä= ãìëí= ÄÉ= êÉãçîÉÇ= éêáçê= íç= ìëÉ= áå= íÜÉä~ÄÉäáåÖ=êÉ~ÅíáçåK

♦ fí= áë= áãéÉê~íáîÉ= íç= éêçíÉÅí= íÜÉ= ok^= Ñêçã= ~åó= Åçåí~ãáå~íáåÖ= ok~ëÉëìåíáä= íÜÉ= Åak^= ëóåíÜÉëáë= áë= ÅçãéäÉíÉK=tÉ~ê= ÑêÉëÜ= ÖäçîÉëI= ìëÉ= åÉïäó~ìíçÅä~îÉÇ= éáéÉí= íáéëI= ~åÇ= ~îçáÇ= ìëáåÖ= éáéÉí= íáéë= çê= ãáÅêçÅÉåíêáÑìÖÉíìÄÉë=íÜ~í=Ü~îÉ=ÄÉÉå=Ü~åÇäÉÇ=ïáíÜçìí=ÖäçîÉëK=oáÄçåìÅäÉ~ëÉ=^=Å~ååçí=ÄÉÇÉëíêçóÉÇ= Äó= åçêã~ä= ~ìíçÅä~îáåÖ= ~äçåÉK= _~âáåÖ= çê=abm`= íêÉ~íãÉåí= áëêÉÅçããÉåÇÉÇK

♦ mçäó^= ãok^= Å~å= ÄÉ= ìëÉÇ= áåëíÉ~Ç= çÑ= íçí~ä= ãok^= áå= íÜáë= éêçíçÅçäK

páãéäó=êÉéä~ÅÉ=íÜÉ=OM=µÖ=çÑ=íçí~ä=ok^=ïáíÜ=MKQÓN=µÖ=çÑ=éçäó^=ãok^K

♦ qÜÉ= ~ãçìåí= çÑ= íçí~ä= ok^= ìëÉÇ= áå= íÜáë= éêçíçÅçä= Å~å= ÄÉ= êÉÇìÅÉÇ= Ñêçã

OM=µÖ=íç=NM=µÖI=ÜçïÉîÉêI=äÉëë=ä~ÄÉäÉÇ=Åak^=ïáää=ÄÉ=ÖÉåÉê~íÉÇK

♦ qÜÉ= èì~äáíó= çÑ= íÜÉ= ÑäìçêÉëÅÉåí= ÇóÉ= áë= ÅêáíáÅ~äK= qÜÉ= ìëÉ= çÑ= é~êíá~ääóÇÉÖê~ÇÉÇ=ÑäìçêÉëÅÉåí=ÇóÉë=ã~ó=êÉëìäí=áå=~=ÜáÖÜÉê=ÑäìçêÉëÅÉåí=Ä~ÅâÖêçìåÇÑçääçïáåÖ= ÜóÄêáÇáò~íáçåK= aç= åçí= êÉëìëéÉåÇ= íÜÉ= kepJÉëíÉê= ÑäìçêÉëÅÉåíÇóÉë=áå=~å=~èìÉçìë=ëçäìíáçå=ëìÅÜ=~ë=ï~íÉêK


♦ qÜÉ= ÑäìçêÉëÅÉåÅÉ= áåíÉåëáíáÉë= çÑ= `v»= PJ= ~åÇ= `v»= RJä~ÄÉäÉÇ= Åak^ÜóÄêáÇáòÉÇ= íç= ãáÅêç~êê~óë= ìåÇÉê= ÅçãéÉíáíáîÉ= ÜóÄêáÇáò~íáçå= ÅçåÇáíáçåëÜ~îÉ= ~= ÜáÖÜ= ÇÉÖêÉÉ= çÑ= ÅçêêÉä~íáçåK= fÑ= çíÜÉê= ÑäìçêÉëÅÉåí= ÇóÉë= é~áêë= ~êÉìëÉÇ= íç= ä~ÄÉä= Åak^I= ïÉ= ÜáÖÜäó= êÉÅçããÉåÇ= íÜ~í= íÜÉ= ë~ãÉ= Åak^= ÄÉä~ÄÉäÉÇ= ïáíÜ= É~ÅÜ= çÑ= íÜÉ= ÑäìçêÉëÅÉåí= ÇóÉëI= ÜóÄêáÇáòÉÇ= íç= ~= ãáÅêç~êê~óìåÇÉê=ÅçãéÉíáíáîÉ=ÜóÄêáÇáò~íáçå=ÅçåÇáíáçåëI=~åÇ=íÜÉ=ÅçêêÉä~íáçå=ÄÉíïÉÉåíÜÉ=ÑäìçêÉëÅÉåÅÉ= áåíÉåëáíáÉë=Å~äÅìä~íÉÇK=qÜÉ= áÇÉ~ä=ÅçêêÉä~íáçå= áë=NKM= ~åÇáåÇáÅ~íÉë=íÜ~í=íÜÉêÉ=áë=~=NWN=ÅçêêÉä~íáçå=ÄÉíïÉÉå=Åak^=ä~ÄÉäÉÇ=ïáíÜ=É~ÅÜçÑ=íÜÉ=ÑäìçêÉëÅÉåí=ÇóÉëK=qÜÉ=ìëÉ=çÑ=ÑäìçêÉëÅÉåí=ÇóÉë=êÉëìäíáåÖ=áå=ä~ÄÉäÉÇÅak^=ïáíÜ= ÜáÖÜ= ÅçêêÉä~íáçå= êÉëìäíë= áå=ãçêÉ= ~ÅÅìê~íÉ= áÇÉåíáÑáÅ~íáçå= çÑÇáÑÑÉêÉåíá~ääó=ÉñéêÉëëÉÇ=ÖÉåÉë=íÜ~å=íÜÉ=ìëÉ=çÑ=ÑäìçêÉëÅÉåí=ÇóÉë=êÉëìäíáåÖáå=ä~ÄÉäÉÇ=Åak^=ïáíÜ=äçï=ÅçêêÉä~íáçåK

♦ qÜÉ= êÉÅçããÉåÇÉÇ=ÄäçÅâáåÖ=ak^= ëçìêÅÉë= ~êÉ= ÉáíÜÉê= Üìã~å= çê=ãçìëÉ`lqJN= ak^∆I= óÉ~ëí= íok^I= ~åÇ= éçäó= ÇE^FQMÓSMK= fÑ= Üìã~å= ak^Åçåí~áåáåÖ=êÉéÉíáíáîÉ=ëÉèìÉåÅÉë=áë=ëéçííÉÇ=çå=íÜÉ=ãáÅêç~êê~óI=ìëÉ=Üìã~å`lqJN= ak^∆= áå= íÜÉ= ÄäçÅâáåÖ= ëçäìíáçåK= eìã~å= `lqJN= ak^∆= áëéä~ÅÉåí~ä=ak^=íÜ~í=áë=RM=íç=PNM=Äé=áå=ëáòÉ=~åÇ=áë=ÉåêáÅÜÉÇ=Ñçê=êÉéÉíáíáîÉak^= ëÉèìÉåÅÉë= ëìÅÜ= ~ë= íÜÉ= ^äì= ~åÇ= héå= Ñ~ãáäóK

2, 3= fÑ= ãçìëÉ= ak^

Åçåí~áåáåÖ=êÉéÉíáíáîÉ=ëÉèìÉåÅÉë=áë=ëéçííÉÇ=çå=íÜÉ=ãáÅêç~êê~óI=ìëÉ=ãçìëÉ`lqJN=ak^∆=áå=íÜÉ=ÄäçÅâáåÖ=ëçäìíáçåK=jçìëÉ=`lqJN=ak^∆=áë=ãçìëÉak^= íÜ~í= áë= RM= íç= PMM= Äé= áå= ëáòÉ= ~åÇ= áë= ÉåêáÅÜÉÇ= Ñçê= êÉéÉíáíáîÉ=ak^ëÉèìÉåÅÉë= ëìÅÜ= ~ë= íÜÉ= _NI= _OI= ~åÇ= iN= Ñ~ãáäó= ãÉãÄÉêëK

4, 5= fåÅäìÇáåÖ

Üìã~å= çê= ãçìëÉ= `lqJN= ak^∆= áå= íÜÉ= ÜóÄêáÇáò~íáçå= ãáñíìêÉ= êÉÇìÅÉëìåÇÉëáêÉÇ= ÜóÄêáÇáò~íáçå= ÄÉíïÉÉå= êÉéÉíáíáîÉ= ak^= ëÉèìÉåÅÉë= áå= íÜÉä~ÄÉäÉÇ= Åak^= ~åÇ= éêçÄÉ= ak^K= vÉ~ëí= íok^= êÉÇìÅÉë= ìåÇÉëáêÉÇåçåëéÉÅáÑáÅ=ak^=ÜóÄêáÇáò~íáçåK=mçäó=ÇE^F=íÜ~í=áë=QM=íç=SM=Ç^qm=Ä~ëÉëáå= äÉåÖíÜ= éêçãçíÉë= ëéÉÅáÑáÅ= ÜóÄêáÇáò~íáçå= ÄÉíïÉÉå= íÜÉ= ä~ÄÉäÉÇ= Åak^~åÇ=éêçÄÉ=ak^=Äó=êÉÇìÅáåÖ=ÜóÄêáÇáò~íáçå=çÑ=íÜÉ=éçäó^=ëÉèìÉåÅÉë=áå=íÜÉéêçÄÉ=ak^=íç=íÜÉ=éçäóq=íê~Åí=áå=íÜÉ=ä~ÄÉäÉÇ=Åak^K

♦ qÜÉ=êÉÅçããÉåÇÉÇ=îçäìãÉ=çÑ=ÜóÄêáÇáò~íáçå=ëçäìíáçå=Åçåí~áåáåÖ=ä~ÄÉäÉÇ

Åak^= íç= ìëÉ= Ñçê= éêçÄáåÖ= ak^= ~êê~óë= áë= O= µä= éÉê= ÅãO= çÑ= ÅçîÉêëäáéëìêÑ~ÅÉ=~êÉ~K=qÜáë=îçäìãÉ=çÑ=ÜóÄêáÇáò~íáçå=ëçäìíáçåI=éêÉé~êÉÇ=~ÅÅçêÇáåÖíç= íÜÉ= éêçíçÅçäë= éêçîáÇÉÇI= áë= ëìÑÑáÅáÉåí= ÉîÉå= Ñçê= ÜáÖÜ= ÇÉåëáíó

ãáÅêç~êê~óëK= qÜÉ= Ä~ëáÅ= éêçíçÅçä= éêçÇìÅÉë= OM= µä= çÑ= ÜóÄêáÇáò~íáçåëçäìíáçåI= ïÜáÅÜ= áë= ëìÑÑáÅáÉåí= Ñçê= ~= íçí~ä= ãáÅêç~êê~ó= ëìêÑ~ÅÉ= ~êÉ~= çÑ

NM=ÅãOK=fåÅêÉ~ëáåÖ=íÜÉ=îçäìãÉ=çÑ=ÜóÄêáÇáò~íáçå=ëçäìíáçå=íç=Q=µä=éÉê=ÅãO

ã~ó=Ñ~Åáäáí~íÉ=ÅçîÉêëäáé=ã~åáéìä~íáçå=áå=ëçãÉ=Å~ëÉëK=^ÇÇáíáçå~ä=îçäìãÉáåÅêÉ~ëÉë=~êÉ=åçí= êÉÅçããÉåÇÉÇI=ÜçïÉîÉêI= ëáåÅÉ=ÜáÖÜ= ëçäìíáçå=îçäìãÉëã~ó= Å~ìëÉ= íÜÉ= ÅçîÉêëäáé= íç= ÇêáÑí= çå= íÜÉ= ëäáÇÉ= ëìêÑ~ÅÉ= ÇìêáåÖÜóÄêáÇáò~íáçåK=píê~í~ÖÉåÉ=ÇçÉë=åçí=êÉÅçããÉåÇ=ÇÉÅêÉ~ëáåÖ=íÜÉ=îçäìãÉ=çÑ

ÜóÄêáÇáò~íáçå=ëçäìíáçå=ÄÉäçï=O=µä=éÉê=ÅãO=çÑ=ÅçîÉêëäáé=ëìêÑ~ÅÉ=~êÉ~K

♦ qÜÉ=Ä~ëáÅ=éêçíçÅçäë=éêçîáÇÉÇ=ïáää=êÉëìäí=áå=íÜÉ=çéíáã~ä=ÅçåÅÉåíê~íáçå=çÑÅak^= ÇìêáåÖ= ÜóÄêáÇáò~íáçå= Ñçê= ãçëí= ~ééäáÅ~íáçåëK= ^ééêçéêá~íÉ~ÇàìëíãÉåíë=áå=íÜÉ=Åak^=ÅçåÅÉåíê~íáçå=Ñçê=ëéÉÅá~ä=~ééäáÅ~íáçåë=ãìëí=ÄÉÇÉíÉêãáåÉÇ=ÉñéÉêáãÉåí~ääóK


PROTOCOL

cDNA GenerationmêÉé~êÉ=ëÉé~ê~íÉ=Åak^=ä~ÄÉäáåÖ=êÉ~Åíáçåë=Ñçê=É~ÅÜ=ÑäìçêÉëÅÉåí=ÇóÉ=óçì=ïáëÜíç= ìëÉK= qÜáë= éêçíçÅçä= éêçÇìÅÉë= ~= ëìÑÑáÅáÉåí= ~ãçìåí= çÑ= ä~ÄÉäÉÇ= Åak^= éÉêêÉ~Åíáçå= íç= ÜóÄêáÇáòÉ= íç= ~= íçí~ä= ãáÅêç~êê~ó= ëìêÑ~ÅÉ= ~êÉ~= çÑ= ~ééêçñáã~íÉäóNM=ÅãOK

==NK cçê=É~ÅÜ=ä~ÄÉäáåÖ=êÉ~ÅíáçåI=êÉëìëéÉåÇ=OM=µÖ=çÑ=íçí~ä=ok^=áå=NOKM=µä=çÑabm`=ï~íÉêK

Note fÑ= íÜÉ= ~ÇÇáíáçå= çÑ= Åçåíêçä= ãok^= áë= ÇÉëáêÉÇI= Ñçê= Éñ~ãéäÉ= íçÖ~ìÖÉ=íÜÉ=ëìÅÅÉëë=çÑ=íÜÉ=ä~ÄÉäáåÖ=êÉ~ÅíáçåI=~ÇÇ=áí=~í=íÜáë=ëíÉéI

Äìí=ÉåëìêÉ=íÜ~í=íÜÉ=Ñáå~ä=îçäìãÉ=~í=íÜÉ=ÉåÇ=çÑ=ëíÉé=N=áë=NOKM=µäK

==OK ^ÇÇ=N=µä=çÑ=RMM=åÖLµä= çäáÖçåìÅäÉçíáÇÉ=ÇEqFNOÓNUK= fåÅìÄ~íÉ= ~í= TMø`= ÑçêNM=ãáåìíÉëK=`ççä=çå=áÅÉ=ìåíáä=êÉ~Çó=Ñçê=ìëÉK

==PK `çãÄáåÉ= íÜÉ= ÑçääçïáåÖ= ÅçãéçåÉåíë= áå= ~= ëíÉêáäÉI= ok~ëÉLak~ëÉJÑêÉÉãáÅêçÅÉåíêáÑìÖÉ=íìÄÉW

O=µä=çÑ=NM�=píê~í~pÅêáéí=êÉ~Åíáçå=ÄìÑÑÉêN=µä=çÑ=OM�=Çkqm=ãáñ

NKR=µä=çÑ=MKN=j=aqq

MKR=µä=çÑ=ok~ëÉ=_äçÅâ=EQM=rLµäF

==QK ^ÇÇ=íÜÉ=~ååÉ~äÉÇ=éêáãÉê=~åÇ=ok^=EÑêçã=ëíÉé=OF=íç=íÜÉ=ãáñíìêÉK

==RK ^ÇÇ= N= µä= çÑ= RM= rLµä= píê~í~pÅêáéí= oq= ~åÇ= áåÅìÄ~íÉ= ~í= QUø`= ÑçêOR=ãáåìíÉëK

==SK ^ÇÇ=~åçíÜÉê=N=µä=çÑ=RM=rLµä=píê~í~pÅêáéí=oq=~åÇ=áåÅìÄ~íÉ=~í=QUø`=Ñçê~å=~ÇÇáíáçå~ä=PR=ãáåìíÉëK

==TK ^ÇÇ= NM= µä= çÑ= N= j= k~le= ~åÇ= áåÅìÄ~íÉ= ~í= TMø`= Ñçê= NM= ãáåìíÉë= íçÜóÇêçäóòÉ=ok^K

==UK `ççä=íç=êççã=íÉãéÉê~íìêÉ=ëäçïäóX=Çç=åçí=Åççä=çå=áÅÉK

==VK péáå=íìÄÉ=ÄêáÉÑäó=íç=ÅçääÉÅí=ÅçåíÉåíëK

NMK ^ÇÇ=NM=µä=çÑ=N=j=e`ä=íç=åÉìíê~äáòÉ=íÜÉ=ëçäìíáçåK


cDNA PurificationqÜÉ= Åak^=ãìëí= ÄÉ= éìêáÑáÉÇ= íç= êÉãçîÉ= ìåáåÅçêéçê~íÉÇ= åìÅäÉçíáÇÉëI= ÄìÑÑÉêÅçãéçåÉåíë= ~åÇ= ÜóÇêçäóòÉÇ= ok^K= píê~í~ÖÉåÉ= êÉÅçããÉåÇë= ~å= ÉíÜ~åçäéêÉÅáéáí~íáçå=íç=éìêáÑó=íÜÉ=Åak^K=fåÅçãéäÉíÉ=êÉãçî~ä=çÑ=íÜÉ=qêáë=~åÇ=bíleïáää=êÉëìäí=áå=äçïÉê=~ãáåç=~ääóäJÇóÉ=ÅçìéäáåÖ=ÉÑÑáÅáÉåÅóK=`~êÉ=ãìëí=ÄÉ=í~âÉåíç=ÉåëìêÉ= íÜ~í=íÜÉ=éÉääÉí= áë=ÅçãéäÉíÉäó=Çêó=E~í= íÜÉ=ÉåÇ=çÑ= ëíÉé=SF= áåÇáÅ~íáåÖÅçãéäÉíÉ= êÉãçî~ä= çÑ= íÜÉ= bíle= ÄÉÑçêÉ= éêçÅÉÉÇáåÖ= íç= íÜÉ= ÇóÉ= ÅçìéäáåÖêÉ~ÅíáçåK

NK ^ÇÇ=Q=µä=çÑ=P=j=ëçÇáìã=~ÅÉí~íÉI=ée=QKR=íç=íÜÉ=êÉ~ÅíáçåK

OK ^ÇÇ=N=µä=çÑ=OM=ãÖLãä=ÖäóÅçÖÉå=íç=íÜÉ=êÉ~ÅíáçåK

PK ^ÇÇ=NMM=µä=çÑ=áÅÉJÅçäÇ=VRB=bíleK

QK fåÅìÄ~íÉ=~í=ÓOMø`=Ñçê=~í=äÉ~ëí=PM=ãáåìíÉëK=qÜÉ=êÉ~Åíáçå=Å~å=ÄÉ=ëíçêÉÇ=~ííÜáë=éçáåí=Ñçê=ëÉîÉê~ä=Ç~óë=çê=ìé=íç=O=ãçåíÜëK

RK péáå= íÜÉ= êÉ~Åíáçå= ëíçêÉÇ= áå= bíle= ïáíÜ= k~l^Å= ~í= NPÓNQIMMM=�=Ö= ÑçêNR=ãáåìíÉë=~í=Qø`K=`~êÉÑìääó=ÇÉÅ~åí=ëìéÉêå~í~åíK

SK t~ëÜ=ïáíÜ=MKR=ãä= áÅÉ= ÅçäÇ= TMB=bíle=~åÇ= ëéáå= ~í= NPÓNQIMMM=�=Ö= ÑçêNR=ãáåìíÉë= ~í= Qø`K=`~êÉÑìääó=ÇÉÅ~åí= ëìéÉêå~í~åí= ~åÇ= ~ääçï= íç= ~áê= ÇêóK^= î~Åììã= ÇêóÉê= Å~å= ÄÉ= ìëÉÇ= íç= ëéÉÉÇ= ìé= íÜÉ= éêçÅÉëë= Äìíal=klq=çîÉêÇêóK

fÑ= ÅçìéäáåÖ= íç= kepJÉëíÉê= Åçåí~áåáåÖ= ÇóÉë= Ñêçã= ^ãÉêëÜ~ã= mÜ~êã~Åá~_áçíÉÅÜ=EëÉÉ=^ÇÇáíáçå~ä=j~íÉêá~äë=oÉèìáêÉÇF=áë=ÇÉëáêÉÇI=Ñçääçï=íÜÉ=éêçíçÅçäÄÉäçï=EkepJbëíÉê=`çåí~áåáåÖ=aóÉ=`çìéäáåÖ=oÉ~Åíáçå=~åÇ=mìêáÑáÅ~íáçåF= íçÅçìéäÉ=íÜÉ=ÇóÉ=íç=íÜÉ=ãçÇáÑáÉÇ=Åak^K

fÑ= ÅçìéäáåÖ= íç= pqmJÉëíÉê= Åçåí~áåáåÖ= ÇóÉë= Ñêçã= jçäÉÅìä~ê= mêçÄÉë= EëÉÉ^ÇÇáíáçå~ä=j~íÉêá~äë=oÉèìáêÉÇF=áë=ÇÉëáêÉÇI=Ñçääçï=íÜÉ=ìëÉêÛë=ÖìáÇÉ=éêçîáÇÉÇïáíÜ=íÜÉ=ÇóÉ=íç=Å~êêó=çìí=íÜáë=êÉ~ÅíáçåK=rëÉ=íÜÉ=êÉ~ÖÉåíë=éêçîáÇÉÇ=ïáíÜ=íÜ~íâáí= íç= êÉëìëéÉåÇI= ÅçìéäÉ= ~åÇ= éìêáÑó= íÜÉ= Åak^K= cçääçïáåÖ= ÅçìéäáåÖ= ~åÇÅak^= éìêáÑáÅ~íáçåI= éêçÅÉÉÇ= íç= ^å~äóëáë= çÑ= cäìçêÉëÅÉåÅÉJä~ÄÉäÉÇ= Åak^EoÉÅçããÉåÇÉÇFK


NHS-Ester Containing Dye Coupling Reaction (For Use with AmershamPharmacia Biotech Dyes)

Note píê~í~ÖÉåÉ= ÇçÉë= åçí= êÉÅçããÉåÇ= ëÅ~äáåÖJÇçïå= íÜÉ= ÑçääçïáåÖ= ÇóÉÅçìéäáåÖ=éêçíçÅçäK=mÉêÑçêãáåÖ=íÜÉ=êÉ~Åíáçå=ìëáåÖ=êÉÇìÅÉÇ=îçäìãÉëêÉëìäíë=áå=~=ëáÖåáÑáÅ~åíäó=êÉÇìÅÉÇ=ÅçìéäáåÖ=ÉÑÑáÅáÉåÅóK

NK oÉëìëéÉåÇ= Åak^= éÉääÉí= áå= R=µä= çÑ= O�= ÅçìéäáåÖ= ÄìÑÑÉêK= fÑ= éÉääÉí= ï~ëçîÉêÇêáÉÇ=áí=ïáää=ÄÉ=ÇáÑÑáÅìäí=íç=ÖÉí=íÜÉ=éÉääÉí=Ä~Åâ=áåíç=ëçäìíáçåK=dÉåíäóÜÉ~í=~í=PTø`=Ñçê=NR=ãáåìíÉë=íç=~áÇ=áå=íÜÉ=êÉëìëéÉåëáçå=éêçÅÉëëK

OK qÜÉ=Ñáêëí=íáãÉ=~=íìÄÉ=çÑ=ÇóÉ=áë=ìëÉÇI=êÉëìëéÉåÇ=áå=QR=µä=ajplK=rëÉ=íÜÉÜáÖÜJéìêáíó= ajpl= éêçîáÇÉÇ= áå= íÜÉ= âáíK= aç= åçí= ëìÄëíáíìíÉ= ~åçíÜÉêajplK= sçêíÉñ= ÖÉåíäó= íç= ÉåëìêÉ= íÜÉ= éÉääÉí= áë= ÅçãéäÉíÉäó= ëçäìÄáäáòÉÇKqÜÉ=ìåìëÉÇ=ÇóÉ=Å~å=~äáèìçíÉÇ=áå=ëáåÖäÉ=ìëÉ=~äáèìçíë=~åÇ=ëíçêÉÇ=~í=ÓOMø`áå=íÜÉ=Ç~êâ=Ñçê=ëÉîÉê~ä=ãçåíÜëK

Note ajpl=áë=ÜóÖêçëÅçéáÅ=~åÇ=ïáää=~ÄëçêÄ=ãçáëíìêÉ=Ñêçã=íÜÉ=~áêKt~íÉê= ~ÄëçêÄÉÇ= Ñêçã= íÜÉ= ~áê= ïáää= êÉ~Åí= ïáíÜ= íÜÉ= kep= ÉëíÉêéçêíáçå= çÑ= íÜÉ= ÇóÉ= ~åÇ= ëáÖåáÑáÅ~åíäó= êÉÇìÅÉ= çê= Éäáãáå~íÉÇóÉWÅak^= ÅçìéäáåÖ= ÉÑÑáÅáÉåÅóK= qç= êÉÇìÅÉ= ~ÄëçêéíáçåI= ~ääçïíÜÉ=ÇóÉ=íç=êÉ~ÅÜ=êççã=íÉãéÉê~íìêÉ=ÄÉÑçêÉ=çéÉåáåÖ=~åÇ=ëíçêÉíÜÉ=ajpl=~í=êççã=íÉãéÉê~íìêÉK=aç=åçí=äÉ~îÉ=ÉáíÜÉê=íÜÉ=ÇóÉçê=ajpl=ìåÅ~ééÉÇ=ïÜÉå=åçí=áå=ìëÉK=aìêáåÖ=ëíçê~ÖÉI=íáÖÜíäóÅ~é=íÜÉ=êÉëìëéÉåÇÉÇ=ÇóÉ=~åÇ=ëíçêÉ=~í=ÓOMø`=áå=íÜÉ=Ç~êâK

PK ^ÇÇ=R=µä=çÑ=ÇóÉ=íç=íÜÉ=Åak^K=fÑ=íÜÉ=ÇóÉ=ï~ë=ëíçêÉÇ=~í=ÓOMø`=éêáçê=íçìëÉI= ~ääçï= íÜÉ= ÇóÉ= íç= êÉ~ÅÜ= êççã= íÉãéÉê~íìêÉ= ÄÉÑçêÉ= çéÉåáåÖ= íÜÉÅçåí~áåÉêK

QK jáñ=Äó=ÖÉåíäó=éáéÉííáåÖ=ìé=~åÇ=ÇçïåK

RK fåÅìÄ~íÉ=Ñçê=PM=ãáåìíÉë=~í=êççã=íÉãé=áå=íÜÉ=Ç~êâK


Dye-Coupled cDNA PurificationqÜÉ= ÑçääçïáåÖ=ÇóÉJÅçìéäÉÇ=Åak^=éìêáÑáÅ~íáçå=ãÉíÜçÇ=Ü~ë=ÄÉÉå=çéíáãáòÉÇÑçê=ìëÉ=ïáíÜ=íÜÉ=ak^JÄáåÇáåÖ=ëçäìíáçå=~åÇ=ãáÅêçëéáå=Åìéë=éêçîáÇÉÇ=áå=íÜáëâáíK=_êáÉÑäóI= áå= íÜÉ= éêÉëÉåÅÉ= çÑ= ~= ÅÜ~çíêçéáÅ= ë~äí= EáåíêçÇìÅÉÇ= Äó= íÜÉ=ak^JÄáåÇáåÖ=ëçäìíáçåI= áåÅäìÇÉÇ= áå= íÜáë=âáíFI= íÜÉ=ÇóÉJÅçìéäÉÇ=Åak^=ÄáåÇë= íç= íÜÉëáäáÅ~JÄ~ëÉÇ=ÑáÄÉê=ã~íêáñ=ëÉ~íÉÇ=áåëáÇÉ=íÜÉ=ãáÅêçëéáå=ÅìéK=t~ëÜáåÖ=ëíÉéë=~êÉÉãéäçóÉÇ= íç= êÉãçîÉ= ÄìÑÑÉê= ë~äíë= ~åÇ= ìåÅçìéäÉÇ= ÑäìçêÉëÅÉåí= ÇóÉ= Ñêçã= íÜÉÄçìåÇ=Åak^K=cáå~ääó=íÜÉ=Åak^=áë=ÉäìíÉÇ=Ñêçã=íÜÉ=ã~íêáñ=ìëáåÖ=~=äçïJáçåáÅëíêÉåÖíÜ= ëçäìíáçåK= rëÉ= çÑ= íÜÉ= ak^JÄáåÇáåÖ= ëçäìíáçå= ~åÇ= ãáÅêçëéáå= ÅìéëéêçîáÇÉÇ=áå=íÜáë=âáí= áå=ÅçåàìåÅíáçå=ïáíÜ=~äíÉêå~íáîÉ=éìêáÑáÅ~íáçå=éêçíçÅçäë=áëåçí=êÉÅçããÉåÇÉÇK

NK ^ÇÇ= VM= µä= çÑ= N�= qb= EëÉÉ= mêÉé~ê~íáçå= çÑ= oÉ~ÖÉåíëF= íç= íÜÉ= ä~ÄÉäÉÇÅak^K

OK `çãÄáåÉ= NMM= µä= çÑ= ak^= ÄáåÇáåÖ= ëçäìíáçå= ~åÇ= NMM= µä= çÑTMB= EîLîF= bíleK= jáñ= ïÉää= Äó= îçêíÉñáåÖK= j~âÉ= ëìêÉ= íÜ~í= íÜÉ= íïçëçäìíáçåë=~êÉ=ïÉää=ãáñÉÇ=éêáçê=íç=ìëÉK

PK ^ÇÇ=íÜÉ=ak^JÄáåÇáåÖ=ëçäìíáçå=~åÇ=bíle=ãáñíìêÉ=íç=íÜÉ=ä~ÄÉäÉÇ=Åak^~åÇ=ãáñ=Äó=îçêíÉñáåÖK

QK rëáåÖ=~=éáéÉíI=íê~åëÑÉê=íÜÉ=ãáñíìêÉ=íç=~=ãáÅêçëéáå=Åìé=íÜ~í=áë=ëÉ~íÉÇ=áå=~OJãä= êÉÅÉéí~ÅäÉ= íìÄÉK= EbñÉêÅáëÉ= Å~ìíáçå= íç= ~îçáÇ= Ç~ã~ÖáåÖ= íÜÉ= ÑáÄÉêã~íêáñ=ïáíÜ=íÜÉ=éáéÉí=íáéKF=på~é=íÜÉ=Å~é=çÑ=íÜÉ=OJãä=êÉÅÉéí~ÅäÉ=íìÄÉ=çåíçíÜÉ=íçé=çÑ=íÜÉ=ãáÅêçëéáå=ÅìéK

Note qç=ÉåëìêÉ=éêçéÉê=ë~ãéäÉ=ÑäçïI=ìëÉ=íÜÉ=êÉÅÉéí~ÅäÉ=íìÄÉ=íÜ~í=áëéêçîáÇÉÇ=ïáíÜ=íÜÉ=ãáÅêçëéáå=ÅìéëK=aç=åçí= ëìÄëíáíìíÉ=~åçíÜÉêíìÄÉK

RK péáå=íÜÉ=íìÄÉ=áå=~=ãáÅêçÅÉåíêáÑìÖÉ=~í=ã~ñáãìã=ëéÉÉÇ=Ñçê=PM=ëÉÅçåÇëK

Note qÜÉ= ä~ÄÉäÉÇ= Åak^= áë= êÉí~áåÉÇ= áå= íÜÉ= ÑáÄÉê= ã~íêáñ= çÑ= íÜÉãáÅêçëéáå=ÅìéK

SK léÉå= íÜÉ= Å~é= çÑ= íÜÉ= OJãä= êÉÅÉéí~ÅäÉ= íìÄÉI= êÉãçîÉ= ~åÇ= êÉí~áå= íÜÉãáÅêçëéáå= ÅìéI= ~åÇ= ÇáëÅ~êÇ= íÜÉ= ak^JÄáåÇáåÖ= ëçäìíáçå= Åçåí~áåáåÖ= íÜÉìåÅçìéäÉÇ=ÇóÉK

TK `çãÄáåÉ=NMM=µä=çÑ=íÜÉ=ak^=ÄáåÇáåÖ=ëçäìíáçå=~åÇ=NMM=µä=çÑ=TMB=EîLîFbíleK=jáñ=ïÉää=Äó=îçêíÉñáåÖK=j~âÉ=ëìêÉ=íÜ~í=íÜÉ=íïç=ëçäìíáçåë=~êÉ=ïÉääãáñÉÇ=éêáçê=íç=ìëÉK

UK ^ÇÇ= íÜÉ= ak^JÄáåÇáåÖ= ëçäìíáçå= ~åÇ= bíle= ãáñíìêÉ= íç= íÜÉ= ãáÅêçëéáåÅìéK=på~é=íÜÉ=Å~é=çÑ=íÜÉ=êÉÅÉéí~ÅäÉ=íìÄÉ=çåíç=íÜÉ=íçé=çÑ=íÜÉ=ãáÅêçëéáåÅìéK

VK péáå=íÜÉ=íìÄÉ=áå=~=ãáÅêçÅÉåíêáÑìÖÉ=~í=ã~ñáãìã=ëéÉÉÇ=Ñçê=PM=ëÉÅçåÇëK


NMK léÉå= íÜÉ= Å~é= çÑ= íÜÉ= OJãä= êÉÅÉéí~ÅäÉ= íìÄÉI= êÉãçîÉ= ~åÇ= êÉí~áå= íÜÉãáÅêçëéáå=ÅìéI=~åÇ=ÇáëÅ~êÇ=íÜÉ=ak^JÄáåÇáåÖ=ëçäìíáçåLbíle=ãáñíìêÉK

NNK ^ÇÇ=TRM=µä=çÑ=TRB=ÉíÜ~åçä=íç=íÜÉ=ãáÅêçëéáå=Åìé=EëÉÉ=�jáÅêçëéáå=`ìét~ëÜ= _ìÑÑÉêÒ= áå= mêÉé~ê~íáçå= çÑ= oÉ~ÖÉåíëFK= på~é= íÜÉ= Å~é= çÑ= íÜÉêÉÅÉéí~ÅäÉ=íìÄÉ=çåíç=íÜÉ=íçé=çÑ=íÜÉ=ãáÅêçëéáå=ÅìéK

NOK péáå=íÜÉ=íìÄÉ=áå=~=ãáÅêçÅÉåíêáÑìÖÉ=~í=ã~ñáãìã=ëéÉÉÇ=Ñçê=PM=ëÉÅçåÇëK

NPK léÉå= íÜÉ= Å~é= çÑ= íÜÉ= OJãä= êÉÅÉéí~ÅäÉ= íìÄÉI= êÉãçîÉ= ~åÇ= êÉí~áå= íÜÉãáÅêçëéáå=ÅìéI=~åÇ=ÇáëÅ~êÇ=íÜÉ=ï~ëÜ=ÄìÑÑÉêK

NQK oÉéÉ~í=ëíÉéë=NNÓNPK

NRK mä~ÅÉ=íÜÉ=ãáÅêçëéáå=Åìé=Ä~Åâ=áå=íÜÉ=OJãä=êÉÅÉéí~ÅäÉ=íìÄÉ=~åÇ=ëå~é=íÜÉÅ~é=çÑ=íÜÉ=êÉÅÉéí~ÅäÉ=íìÄÉ=çåíç=íÜÉ=ãáÅêçëéáå=ÅìéK

NSK péáå=íÜÉ=íìÄÉ=áå=~=ãáÅêçÅÉåíêáÑìÖÉ=~í=ã~ñáãìã=ëéÉÉÇ=Ñçê=PM=ëÉÅçåÇëKlå=êÉãçî~ä=Ñêçã=íÜÉ=ÅÉåíêáÑìÖÉI=ã~âÉ=ëìêÉ=íÜ~í=~ää=çÑ=íÜÉ=ï~ëÜ=ÄìÑÑÉê=áëêÉãçîÉÇ=Ñêçã=íÜÉ=ãáÅêçëéáå=ÅìéK

NTK qê~åëÑÉê=íÜÉ=ãáÅêçëéáå=Åìé=íç=~=ÑêÉëÜ=NKRJãä=ãáÅêçÅÉåíêáÑìÖÉ=íìÄÉll=~åÇÇáëÅ~êÇ=íÜÉ=OJãä=êÉÅÉéí~ÅäÉ=íìÄÉK

NUK ^ÇÇ=RM=µä=çÑ=NM=ãj=qêáë=Ä~ëÉI=ée=UKR=ÇáêÉÅíäó=çåíç=íÜÉ=íçé=çÑ=íÜÉ=ÑáÄÉêã~íêáñ=~í=íÜÉ=Äçííçã=çÑ=íÜÉ=ãáÅêçëéáå=Åìé=EëÉÉ=�jáÅêçëéáå=`ìé=bäìíáçå_ìÑÑÉêÒ=áå=mêÉé~ê~íáçå=çÑ=jÉÇá~=~åÇ=oÉ~ÖÉåíëFK

NVK fåÅìÄ~íÉ=íÜÉ=íìÄÉ=~í=êççã=íÉãéÉê~íìêÉ=Ñçê=R=ãáåìíÉëK

Note j~ñáãìã=êÉÅçîÉêó=çÑ= íÜÉ= ä~ÄÉäÉÇ=Åak^=Ñêçã=íÜÉ=ãáÅêçëéáåÅìé=ÇÉéÉåÇë=çå=íÜÉ=éeI=íÜÉ=áçåáÅ=ëíêÉåÖíÜI=~åÇ=íÜÉ=îçäìãÉ=çÑíÜÉ=Éäìíáçå=ÄìÑÑÉê=~ÇÇÉÇ=íç=íÜÉ=ãáÅêçëéáå=ÅìéX=íÜÉ=éä~ÅÉãÉåíçÑ= íÜÉ= Éäìíáçå= ÄìÑÑÉê= áåíç= íÜÉ= ãáÅêçëéáå= ÅìéX= ~åÇ= íÜÉáåÅìÄ~íáçå= íáãÉK= j~ñáãìã= êÉÅçîÉêó= áë= çÄí~áåÉÇ= ïÜÉå= íÜÉ

Éäìíáçå=ÄìÑÑÉê=áë=≤NM=ãj=áå=ÅçåÅÉåíê~íáçå=ïáíÜ=ée=TÓVI=ïÜÉå

åçí=äÉëë=íÜ~å=RM=µä=çÑ=Éäìíáçå=ÄìÑÑÉê=áë=~ÇÇÉÇ=ÇáêÉÅíäó=çåíç=íÜÉÑáÄÉê=ã~íêáñ=~í=íÜÉ=Äçííçã=çÑ=íÜÉ=ãáÅêçëéáå=ÅìéI=~åÇ=ïÜÉå=íÜÉíìÄÉ=áë=áåÅìÄ~íÉÇ=Ñçê=R=ãáåìíÉëK

OMK på~é=íÜÉ=Å~é=çÑ=íÜÉ=NKRJãä=ãáÅêçÅÉåíêáÑìÖÉ=íìÄÉ=çåíç=íÜÉ=ãáÅêçëéáå=Åìé~åÇ= ëéáå= íÜÉ= íìÄÉ= áå= ~= ãáÅêçÅÉåíêáÑìÖÉ= ~í= ã~ñáãìã= ëéÉÉÇ= ÑçêPM=ëÉÅçåÇëK

ONK léÉå=íÜÉ=äáÇ=çÑ=íÜÉ=ãáÅêçÅÉåíêáÑìÖÉ=íìÄÉ=~åÇ=êÉÅçîÉê=íÜÉ=Ñäçï=íÜêçìÖÜÅçåí~áåáåÖ=íÜÉ=éìêáÑáÉÇ=ä~ÄÉäÉÇ=Åak^K

ää NKRJãä=Ñä~í=ëå~é=Å~é=ãáÅêçÅÉåíêáÑìÖÉ=íìÄÉë=Ñêçã=`çåíáåÉåí~ä=i~Äçê~íçêó=mêçÇìÅíëI=fåÅK=~êÉ

êÉÅçããÉåÇÉÇK


OOK bäìíÉ=~ÇÇáíáçå~ä=ä~ÄÉäÉÇ=Åak^=Äó=éáéÉííáåÖ=íÜÉ=Ñäçï=íÜêçìÖÜ=Ä~Åâ=çåíçíÜÉ=ÑáÄÉê=ã~íêáñ=çÑ=íÜÉ=ë~ãÉ=ãáÅêçëéáå=ÅìéK

OPK oÉJëÉ~í= íÜÉ=ëéáå= Åìé=çå= íÜÉ= ë~ãÉ=OJãä= êÉÅÉéí~ÅäÉ= íìÄÉ= íÜ~í= Åçåí~áåÉÇíÜÉ=äáèìáÇ=Ñêçã=íÜÉ=ÑáêëíJé~ëë=ÉäìíáçåK

OQK fåÅìÄ~íÉ=íÜÉ=íìÄÉ=~í=êççã=íÉãéÉê~íìêÉ=Ñçê=R=ãáåìíÉëK

ORK på~é=íÜÉ=Å~é=çÑ=íÜÉ=NKRJãä=ãáÅêçÅÉåíêáÑìÖÉ=íìÄÉ=çåíç=íÜÉ=ãáÅêçëéáå=Åìé~åÇ= ëéáå= íÜÉ= íìÄÉ= áå= ~= ãáÅêçÅÉåíêáÑìÖÉ= ~í= ã~ñáãìã= ëéÉÉÇ= ÑçêPM=ëÉÅçåÇëK

OSK léÉå=íÜÉ=äáÇ=çÑ=íÜÉ=ãáÅêçÅÉåíêáÑìÖÉ=íìÄÉ=~åÇ=êÉÅçîÉê=íÜÉ=ÑäçïJíÜêçìÖÜÅçåí~áåáåÖ=íÜÉ=éìêáÑáÉÇ=ä~ÄÉäÉÇ=Åak^K

OTK e~êîÉëí= çåÉ= Ñáå~ä= Éäìíáçå= Ñêçã= íÜÉ= ãáÅêçëéáå= Åìé= Äó= êÉéÉ~íáåÖëíÉéë=OOÓOSK

OUK léÉå=íÜÉ=äáÇ=çÑ=íÜÉ=ãáÅêçÅÉåíêáÑìÖÉ=íìÄÉ=~åÇ=êÉÅçîÉê=íÜÉ=Ñäçï=íÜêçìÖÜÅçåí~áåáåÖ=íÜÉ=éìêáÑáÉÇ=ä~ÄÉäÉÇ=Åak^K

OVK qç=êÉÇìÅÉ=íÜÉ=îçäìãÉ=çÑ=íÜÉ=ä~ÄÉäÉÇ=Åak^I=éä~ÅÉ=íÜÉ=ä~ÄÉäÉÇ=Åak^=áå~=ÅÉåíêáÑìÖÉ=~åÇ=~ééäó=~=î~ÅììãK=`çåíáåìÉ=íç=~ééäó=~=î~Åììã=ìåíáä=íÜÉ

îçäìãÉ=Ü~ë=ÄÉÉå=êÉÇìÅÉÇ=íç=~ééêçñáã~íÉäó=NQ=µäK

Note fÑ= ~= ÜáÖÜÉê= ÅçåÅÉåíê~íáçå= çÑ= ä~ÄÉäÉÇ= Åak^= áë= ÇÉëáêÉÇI= íÜÉîçäìãÉ=ã~ó= ÄÉ= êÉÇìÅÉÇ= ÑìêíÜÉê= Äó= ÅçåíáåìÉÇ= ÅÉåíêáÑìÖ~íáçåìåÇÉê=î~ÅììãK=pÅ~äÉ=Ççïå=íÜÉ=îçäìãÉë=çÑ=çíÜÉê=ÜóÄêáÇáò~íáçåëçäìíáçå= ÅçãéçåÉåíë= ~ÅÅçêÇáåÖäó= EëÉÉ= íÜÉ= eóÄêáÇáò~íáçåëÉÅíáçåFK

PMK ^Çàìëí=íÜÉ=îçäìãÉ=çÑ=íÜÉ=ä~ÄÉäÉÇ=Åak^=íç=~=Ñáå~ä=îçäìãÉ=çÑ=NQ=µä=Äó~ÇÇáåÖ=ÇeOlK


Analysis of Fluorescence-labeled cDNA (Recommended)qÜçìÖÜ= íÜÉ= ä~ÄÉäÉÇ= Åak^= ã~ó= ÄÉ= îáëáÄäó= ÅçäçêÉÇ= áí= áë= ÇáÑÑáÅìäí= íçÇáëíáåÖìáëÜ=ïÜÉíÜÉê=íÜÉ=Åçäçê=áë=Ñêçã=ìåêÉ~ÅíÉÇ=ÇóÉ=çê=íÜÉ=ÇóÉ=ÅçìéäÉÇ=íç=íÜÉÅak^K= qÜÉêÉÑçêÉ= píê~í~ÖÉåÉ= êÉÅçããÉåÇë= íÜ~í= íÜÉ= Åak^= ÄÉ= ~å~äóòÉÇ= íçÇÉíÉêãáåÉ=áÑ=íÜÉ=ÑäìçêÉëÅÉåÅÉJä~ÄÉäÉÇ=ÇóÉ=Ü~ë=ÄÉÉå=ÅçìéäÉÇI=áÑ=íÜÉ=ìåÅçìéäÉÇÇóÉ= áë= ÅçãéäÉíÉäó= êÉãçîÉÇI= ~åÇ= áÑ= íÜÉ= ok^= íÉãéä~íÉ= ëíê~åÇ= Ü~ë= ÄÉÉåÅçãéäÉíÉäó=ÜóÇêçäóòÉÇK=`çãéäÉíÉ=êÉãçî~ä=çÑ=íÜÉ=ok^=áë=ÅêáíáÅ~ä=Ñçê=çéíáã~äÜóÄêáÇáò~íáçå=çÑ=íÜÉ=ä~ÄÉäÉÇ=Åak^=~åÇ=éêçÄÉ=ak^K

cçê= íÜáë= ~å~äóëáëI= íÜÉ= Åak^= áë= ÉäÉÅíêçéÜçêÉëÉÇ= çå= ~å= ~Ö~êçëÉ= ÖÉäK= qçÇÉíÉêãáåÉ=áÑ=ÑäìçêÉëÅÉåí=ÇóÉ=Ü~ë=ÄÉÉå=ÅçìéäÉÇI=íÜÉ=Åak^=áë=îáëì~äáòÉÇ=ïáíÜ~= ä~ëÉê= ëÅ~ååÉêK= qç= ÇÉíÉêãáåÉ= áÑ= íÜÉ= ok^= íÉãéä~íÉ= ëíê~åÇ= Ü~ë= ÄÉÉåÅçãéäÉíÉäó=êÉãçîÉÇI=íÜÉ=Åak^=áë=ëí~áåÉÇ=ïáíÜ=ÉíÜáÇáìã=ÄêçãáÇÉ=ÑçääçïáåÖÉäÉÅíêçéÜçêÉëáë=~åÇ=îáëì~äáòÉÇ=ïáíÜ=~=rs=íê~åëáääìãáå~íçêK

NK oÉãçîÉ= MKR= µä= çÑ= É~ÅÜ= ä~ÄÉäÉÇ= Åak^= ~åÇ= ~å~äóòÉ= Äó= ëäáÇÉ= ÖÉäÉäÉÅíêçéÜçêÉëáë= EëÉÉ= ^ééÉåÇáñ= fF= çå= ~= OB= ~Ö~êçëÉ= qêáëJ~ÅÉí~íÉ= ÖÉäKmêÉé~êÉ= `vJä~ÄÉäÉÇ= Åak^= ïáíÜ= ãáåáã~ä= ÇóÉ= äç~ÇáåÖ= ÄìÑÑÉê= EëÉÉmêÉé~ê~íáçå=çÑ=oÉ~ÖÉåíëFK=mêÉé~êÉ=~=ak^=ëáòÉ=ëí~åÇ~êÇ=ìëáåÖ=ëí~åÇ~êÇÇóÉ=äç~ÇáåÖ=ÄìÑÑÉêK

Note qÜÉ=~ÇÇáíáçå=çÑ=ÉíÜáÇáìã=ÄêçãáÇÉ=íç=íÜÉ=ÖÉä=çê=êìååáåÖ=ÄìÑÑÉêïáää=~ÑÑÉÅí=ÇÉíÉÅíáçå=çÑ=íÜÉ=`v=PJ=çê=`v=RJä~ÄÉäÉÇ=ak^=ÇìêáåÖëÅ~ååáåÖK= píê~í~ÖÉåÉ= êÉÅçããÉåÇë= áåÅäìÇáåÖ= ~= ak^= ëáòÉëí~åÇ~êÇ= áå= ~= ëÉé~ê~íÉ= ä~åÉK= ^ÑíÉê= ÉäÉÅíêçéÜçêÉëáëI= ëÉé~ê~íÉíÜáë=ä~åÉ=Ñêçã=íÜÉ=çíÜÉêë=~åÇ=ëí~áå=ïáíÜ=ÉíÜáÇáìã=ÄêçãáÇÉK

OK aÉíÉÅí= `v= RJä~ÄÉäÉÇ= ak^= Äó= ëÅ~ååáåÖ= íÜÉ= ÖÉä= ïáíÜ= ~= SPRJåã= ä~ëÉêïáíÜ=~=TMMJåã=Éãáëëáçå=ÑáäíÉê=~åÇ=`v=PJä~ÄÉäÉÇ=ak^=Äó=ëÅ~ååáåÖ=íÜÉÖÉä= ïáíÜ= ~= RPOJåã= ä~ëÉê= ïáíÜ= ~= RUMJåã= Éãáëëáçå= ÑáäíÉêK= `çãé~êÉ= íçÉíÜáÇáìã= ÄêçãáÇÉÓëí~áåÉÇ= ak^= ëáòÉ= ëí~åÇ~êÇ= íç= ÇÉíÉêãáåÉ= äÉåÖíÜ= çÑä~ÄÉäÉÇ=Åak^K

Note i~ÄÉäÉÇ= ak^= ëÜçìäÇ= ÄÉ= QMMÓNRMM= Äé= áå= äÉåÖíÜK= ^åóìåÅçìéäÉÇ= ÇóÉ= ïáää= ~ééÉ~ê= ~ë= ~= ÄêáÖÜí= Ä~åÇ= ~í= íÜÉ= äÉ~ÇáåÖÑêçåí=çÑ=íÜÉ=ÖÉäK

PK pí~áå=íÜÉ=ÖÉä=Åçåí~áåáåÖ=íÜÉ=Åak^=ïáíÜ=ÉíÜáÇáìã=ÄêçãáÇÉ=~åÇ=îáëì~äáòÉïáíÜ=~=rs=íê~åëáääìãáå~íçêK

Note `çãéäÉíÉ= êÉãçî~ä=çÑ= íÜÉ=ok^= íÉãéä~íÉ= áë= áåÇáÅ~íÉÇ= Äó= îÉêóéççê=çê=åç=ëí~áåáåÖ=çÑ=íÜÉ=Åak^=ïáíÜ=ÉíÜáÇáìã=ÄêçãáÇÉK


HybridizationqÜÉ= ak^= ÄäçÅâáåÖ= ~åÇ= ÜóÄêáÇáò~íáçå= ëçäìíáçåë= ëÜçìäÇ= ÄÉ= íÉëíÉÇ= Ñçê~ìíçÑäìçêÉëÅÉåÅÉ=éêáçê=íç=ìëÉ=Äó=ëéçííáåÖ=çå=~=ÅäÉ~å=Öä~ëë=ãáÅêçëÅçéÉ=ëäáÇÉ~åÇ=ëÅ~ååáåÖ=ìëáåÖ=íÜÉ=ãáÅêç~êê~ó=ëÅ~ååÉêK

NK ^ÇÇ=N=µä=çÑ=NM=µÖLµä=Üìã~å=çê=ãçìëÉ=`lqJN=ak^∆I=N=µä=çÑ=U=µÖLµäéçäó=ÇE^FQMÓSMI= ~åÇ= N= µä= çÑ= Q=µÖLµä= óÉ~ëí= íok^= íç= É~ÅÜ= ÅçåÅÉåíê~íÉÇä~ÄÉäÉÇ=Åak^K

OK ^ÇÇ=P=µä=çÑ=OM�=pp`=~åÇ=MKR=µä=NMB=pap=íç=É~ÅÜ=ä~ÄÉäÉÇ=Åak^=Ñçê=~

Ñáå~ä=îçäìãÉ=çÑ=OM=µäK

Note qÜÉ=ãáåáãìã=êÉÅçããÉåÇÉÇ=~ãçìåí=çÑ=ÜóÄêáÇáò~íáçå=ëçäìíáçå

áë=OKM=µä=éÉê=ÅãO=çÑ=ÅçîÉêëäáéK=qÜÉ=ëáòÉ=çÑ=íÜÉ=ÅçîÉêëäáé=ëÜçìäÇÉñÅÉÉÇ= íÜÉ= ëáòÉ= çÑ= íÜÉ= ãáÅêç~êê~ó= Äó= OÓQ=ãã= áå= É~ÅÜÇáãÉåëáçåK=píçêÉ=ìåìëÉÇ=ä~ÄÉäÉÇ=Åak^=~í=Qø`=áå=íÜÉ=Ç~êâ=Ñçêìé=íç=O=ïÉÉâëK

PK oÉãçîÉ= ~å= ~ééêçéêá~íÉ= ~ãçìåí= çÑ= ä~ÄÉäÉÇ= Åak^= EÑêçã= ëíÉé= OF= íçÜóÄêáÇáòÉ= íç= íÜÉ=ãáÅêç~êê~óK=eÉ~í= ~í= VVø`=Ñçê=O=ãáåìíÉëI= íÜÉå=Åççä= íçQRø`K=péáå=Ççïå=ÅçåÇÉåë~íáçåK=fåÅìÄ~íÉ=~í=QRø`=Ñçê=OM=ãáåìíÉë=ÄÉÑçêÉÅçåíáåìáåÖ=íç=ëíÉé=RK

QK qç= áÇÉåíáÑó=íÜÉ=éçëáíáçå=çÑ= íÜÉ=ãáÅêç~êê~ó=çå=~=ëäáÇÉI=~äáÖå=~= íÉãéä~íÉïáíÜ=íÜÉ=ãáÅêç~êê~óK

RK ^ééäó= ä~ÄÉäÉÇ= Åak^=éêÉé~êÉÇ= Ñêçã= íÉëí=ok^= íç=çåÉ=ãáÅêç~êê~ó= ~åÇä~ÄÉäÉÇ= Åak^= éêÉé~êÉÇ= Ñêçã= Åçåíêçä= ok^= íç= ~åçíÜÉê= ãáÅêç~êê~óK_ìÄÄäÉë= ÄÉíïÉÉå= íÜÉ= ÅçîÉêëäáé= ~åÇ= íÜÉ= ãáÅêç~êê~ó= ãìëí= ÄÉ= ~îçáÇÉÇK`~êÉÑìääó=éçé= ä~êÖÉ=ÄìÄÄäÉë= áå= íÜÉ=ÜóÄêáÇáò~íáçå=ëçäìíáçå=ïáíÜ= ~=éáéÉííáé= ÄÉÑçêÉ= éä~ÅáåÖ= ÅçîÉêëäáé= çå= íÜÉ= ãáÅêç~êê~ó= Äìí= Çç= åçí= íçìÅÜ= íÜÉãáÅêç~êê~ó= ïáíÜ= íÜÉ= éáéÉí= íáéK= ^ÇÇ= ÅçîÉêëäáéëK= pã~ää= ÄìÄÄäÉë= ïáääÇáë~ééÉ~ê=ïÜÉå=íÜÉ=ãáÅêç~êê~ó=ÜóÄêáÇáò~íáçå=ÅÜ~ãÄÉê=áë=éä~ÅÉÇ=áå= íÜÉï~íÉê= Ä~íÜK= aç= åçí= ~ííÉãéí= íç= êÉéçëáíáçå= íÜÉ= ÅçîÉêëäáé= çåÅÉ= áí= áë= çåíÜÉ=ãáÅêç~êê~óK

SK ^ÇÇ=PM=µä=çÑ=P�=pp`= EÇáäìíÉÇ=ïáíÜ= íÜÉ=abm`=ï~íÉê=éêçîáÇÉÇF= íç= íÜÉáåÇÉåí~íáçåë= çå= É~ÅÜ= ëáÇÉ= çÑ= íÜÉ= ãáÅêç~êê~ó= ÜóÄêáÇáò~íáçå= ÅÜ~ãÄÉêKmä~ÅÉ= ëäáÇÉë= áå= ëÉé~ê~íÉ= ÅÜ~ãÄÉêë= ~åÇ= ëÉ~ä= ~ÅÅçêÇáåÖ= íç= íÜÉã~åìÑ~ÅíìêÉêÛë= áåëíêìÅíáçåëK=_É=Å~êÉÑìä=åçí= íç=ãçîÉ=ÅçîÉêëäáéK=aç=åçíáåîÉêí=íÜÉ=ÅÜ~ãÄÉêK

TK pìÄãÉêÖÉ= íÜÉ=ÅÜ~ãÄÉêë= áå=~=ï~íÉê=Ä~íÜ=~í= SRø`= Ñçê=NQÓNU=ÜçìêëK=açåçí=ÉñÅÉÉÇ=NU=ÜçìêëK


WashingmêÉé~êÉ=ï~ëÜ=ëçäìíáçåë=ìëáåÖ=ok~ëÉLak~ëÉJÑêÉÉ=ï~íÉê=EpÉÉ=mêÉé~ê~íáçå=çÑoÉ~ÖÉåíëFK= aç= åçí= êÉìëÉ= ï~ëÜ= ëçäìíáçåëK= rëÉ= ~= ÑêÉëÜ= ï~ëÜ= ëçäìíáçå= É~ÅÜíáãÉK=aç=åçí=~ääçï=ëäáÇÉë=íç=Çêó=ÄÉíïÉÉå=ï~ëÜ=ëíÉéëK=qê~åëÑÉê=ëäáÇÉë=ÇáêÉÅíäóÑêçã=çåÉ=ï~ëÜ=ëçäìíáçå=íç=íÜÉ=çíÜÉêK

NK mêÉé~êÉ=RMM=ãä=çÑ=MKO=µãÓÑáäíÉêÉÇ=MKR�=pp`I=MKMNB=pap=EÇáäìíÉÇ=ïáíÜok~ëÉLak~ëÉJÑêÉÉ=eOlF=~åÇ=éä~ÅÉ=áå=~=ÅäÉ~å=O=äáíÉê=ÄÉ~âÉê=EÑáêëí=ï~ëÜëçäìíáçåFK=mêÉé~êÉ=~å=~ÇÇáíáçå~ä=RM=ãä=çÑ=íÜÉ=Ñáêëí=ï~ëÜ=ëçäìíáçå=Ñçê=É~ÅÜëäáÇÉ= ~åÇ= éä~ÅÉ= áå= ëÉé~ê~íÉ= RMJãä= ÅçåáÅ~äëK= mêÉé~êÉ= RMM= ãä= çÑ

MKO=µãÓÑáäíÉêÉÇ=MKMS�=pp`=~åÇ=éä~ÅÉ=áåíç=~=ÅäÉ~å=O=äáíÉê=ÄÉ~âÉê=EëÉÅçåÇï~ëÜ= ëçäìíáçåFK= mä~ÅÉ= ~= ÅäÉ~å= ã~ÖåÉíáÅ= ëíáêÄ~ê= áåíç= É~ÅÜ= ÄÉ~âÉêÅçåí~áåáåÖ=ï~ëÜ=ëçäìíáçåK=RMM=ãä=çÑ=É~ÅÜ=ï~ëÜ=ëçäìíáçå=áë=ëìÑÑáÅáÉåí=Ñçêìé= íç=NM=ëäáÇÉëK= fÑ=ãçêÉ= íÜ~å=NM=ëäáÇÉë=~êÉ=ÄÉáåÖ=ï~ëÜÉÇI= áåÅêÉ~ëÉ= íÜÉï~ëÜ=îçäìãÉ=Äó=RM=ãä=Ñçê=É~ÅÜ=~ÇÇáíáçå~ä=ëäáÇÉK

OK pìëéÉåÇ=~= ëäáÇÉ= Å~êêáÉê= áå= íÜÉ= Ñáêëí=ï~ëÜ= ëçäìíáçå=~ééêçñáã~íÉäó=N= Åã~ÄçîÉ=íÜÉ=ã~ÖåÉíáÅ=ëíáêÄ~êK=j~âÉ=ëìêÉ=íÜ~í=íÜÉ=ëäáÇÉë=ïáää=ÄÉ=ÅçãéäÉíÉäóÅçîÉêÉÇ=ïáíÜ= íÜÉ=ï~ëÜ= ëçäìíáçå=ïÜÉå= éä~ÅÉÇ= áåíç= íÜÉ= ëäáÇÉ= Å~êêáÉêK= fÑåÉÉÇÉÇI=éêÉé~êÉ=~åÇ=~ÇÇ=ãçêÉ=ï~ëÜ=ëçäìíáçåK

PK oÉãçîÉ=ëäáÇÉë=Ñêçã=ÜóÄêáÇáò~íáçå=ÅÜ~ãÄÉêK=aç=åçí=ÉñéçëÉ=ãáÅêç~êê~óíç=~áê=Äó=êÉãçîáåÖ=ÅçîÉêëäáéK

QK mä~ÅÉ= ëäáÇÉë= áåíç= ëÉé~ê~íÉ= ÅçåáÅ~äë= Åçåí~áåáåÖ= RM=ãä= çÑ= íÜÉ= Ñáêëí=ï~ëÜëçäìíáçåK=oÉãçîÉ=ÅçîÉêëäáé=Äó=ÖÉåíäÉ=~Öáí~íáçåK=fÑ=åÉÉÇÉÇI=íÜÉ=ÅçîÉêëäáéÅ~å= ÄÉ= êÉãçîÉÇ= ïáíÜ= ÑçêÅÉéëK= fÑ= ÑçêÅÉéë= ~êÉ= ìëÉÇI= Çç= åçí= íçìÅÜãáÅêç~êê~ó= ïáíÜ= ÑçêÅÉéëK= ^ÑíÉê= êÉãçî~ä= çÑ= íÜÉ= ÅçîÉêëäáéI= íê~åëÑÉê= íÜÉëäáÇÉ= íç= íÜÉ= ëäáÇÉ= Å~êêáÉê= íÜ~í= áë= ëìëéÉåÇÉÇ= áå= íÜÉ= Ñáêëí= ï~ëÜ= ëçäìíáçåKt~ëÜ=~í=êççã=íÉãéÉê~íìêÉ=Ñçê=RÓNM=ãáåìíÉë=~í=êççã=íÉãéÉê~íìêÉ=ïáíÜÖÉåíäÉ=ëïáêäáåÖ=çÑ=íÜÉ=ï~ëÜ=ëçäìíáçåK

RK qê~åëÑÉê= íÜÉ= ëäáÇÉ= Å~êêáÉê= Åçåí~áåáåÖ= íÜÉ= ëäáÇÉë= íç= íÜÉ= ëÉÅçåÇ= ï~ëÜëçäìíáçå=~åÇ=ëìëéÉåÇ=íÜÉ=ëäáÇÉë=~ééêçñáã~íÉäó=N=Åã=~ÄçîÉ= íÜÉ=ëíáêÄ~êKj~âÉ= ëìêÉ= íÜ~í= íÜÉ= ëäáÇÉë= ~êÉ= ÅçîÉêÉÇ= Äó= íÜÉ=ï~ëÜ= ëçäìíáçåK=t~ëÜ= ~íêççã=íÉãé=Ñçê=RÓNM=ãáåìíÉë=ïáíÜ=ÖÉåíäÉ=ëïáêäáåÖ=çÑ=íÜÉ=ï~ëÜ=ëçäìíáçåíç=ÉåëìêÉ=íÜÉ=ÅçãéäÉíÉ=êÉãçî~ä=çÑ=papK

SK aêó= ëäáÇÉë= Äó= ÄäçííáåÖ= íÜÉ= ëäáÇÉ= ÜçäÇÉê= ÄêáÉÑäó= çå= é~éÉê= íçïÉäë= ~åÇëéáååáåÖ=~í=NMMM=�=Ö=Ñçê=NÓO=ãáåìíÉëK=fÑ=íÜÉ=ëäáÇÉ=ÜçäÇÉê=Å~ååçí=ÄÉ=ìëÉÇáå=íÜÉ=ÅÉåíêáÑìÖÉI=íê~åëÑÉê=É~ÅÜ=ëäáÇÉ=íç=~=ëÉé~ê~íÉ=Éãéíó=RMJãä=ÅçåáÅ~äéêáçê=íç=ÅÉåíêáÑìÖ~íáçåK=fÑ=íÜÉ=ëäáÇÉ=Ü~ë=~=Ä~ê=ÅçÇÉI=éä~ÅÉ=íÜÉ=ÉåÇ=çÑ=íÜÉëäáÇÉ=ïáíÜ=íÜÉ=Ä~ê=ÅçÇÉ=~í=íÜÉ=Äçííçã=çÑ=íÜÉ=ÅçåáÅ~äK=aç=åçí=~áê=Çêó=ëäáÇÉK

Hybridization Detectioncçääçï=ã~åìÑ~ÅíìêÉêÛë=êÉÅçããÉåÇ~íáçåë=Ñçê=ìëÉ=çÑ=~=ãáÅêç~êê~ó=ëÅ~ååÉêK


TROUBLESHOOTING

Observation Suggestion

Isolate RNA using a method that removes carbohydrates, lipids and cellular proteins

Purify labeled cDNA to remove uncoupled fluorescent dye

Do not handle slides with bare hands; handle slides by the edges with gloved hands or

forceps

Follow the blocking procedure recommended by the microarray manufacturer for

reducing nonspecific hybridization to the microarrays

Avoid the presence of large bubbles in hybridization solution during hybridization; if

large bubbles are present in the hybridization solution after placing on the microarray,

carefully pop the bubble with a pipet tip but do not touch the microarray with the pipet

tip

Clean microarray hybridization chamber prior to use

Add 3× SSC to hybridization chamber during hybridization to prevent slide from drying

out

Do not remove coverslip prior to placing slide in wash solution

Prepare fresh washing solutions each time

Follow washing procedures correctly; do not allow the microarray slides to dry out until

the very last drying step using centrifugation

Spin slide to dry

High background signal

Verify that DNA blocking reagents (COT-1 DNA®, polyA and yeast tRNA) do not

autofluoresce

Use correct concentration of SSC and SDS during hybridization

Do not move coverslip after placing on the array

Smeared DNA spots

Do not wash above room temperature or below a concentration of 0.06× SSC

Isolate the RNA in the presence of a ribonuclease inhibitor, and ensure all labware and

reagents are free of RNases

Remove any inhibitors of StrataScript RT (SDS, EDTA, guanidinium chloride, formamide,

Na2PO

4, or spermidine) with an additional 70% (v/v) ethanol wash following ethanol

precipitation; remove residual ethanol

Verify the integrity of the RNA by denaturing agarose gel electrophoresis

Verify the incorporation of fluorescent dyes and integrity of the labeled cDNA by agarose

gel electrophoresis

Verify that the RNA template strand was completely removed by hydrolysis

Use correct concentration of SSC and SDS during hybridization

Verify that the hybridization solution was placed on the microarray

Verify that the correct side of the slide was scanned for the microarray

Low signal or none at all

Verify that the correct region of the microarray was scanned

Use a low-ionic-strength (≤10 mM) buffer, pH 8.5 as the elution buffer

Do not add less than 50 µl of elution buffer to the microspin cup

Add the elution buffer directly onto the fiber matrix of the microspin cup to ensure

complete coverage of the membrane

Incubate the tube for 5 minutes after adding the elution buffer prior to centrifugation

Low recovery of the labeled

cDNA from the microspin cups

Make sure that all the wash buffer is removed from the microspin cup before adding the

elution buffer


PREPARATION OF REAGENTS

20× SSC Buffer, high-qualityRNase/DNase-free

NTRKP=Ö=çÑ=k~`ä==UUKO=Ö=çÑ=ëçÇáìã=Åáíê~íÉUMMKM=ãä=çÑ=ok~ëÉLak~ëÉJÑêÉÉ=ï~íÉê^Çàìëí=ée=íç=TKM=ïáíÜ=~=ÑÉï=Çêçéë=çÑ

NM=k=k~le^ÇÇ=ok~ëÉLak~ëÉJÑêÉÉ=ï~íÉê=íç=~

Ñáå~ä=îçäìãÉ=çÑ=N=äáíÉê

Human or Mouse COT-1 DNA®,10 µg/µl

`çåÅÉåíê~íÉ=íÜÉ=N=µÖLµä=ëçäìíáçå=çÑ`lqJN=ak^∆=Äó=NMJÑçäÇ=ìëáåÖ=ÉáíÜÉê=çÑ=íÜÉÑçääçïáåÖ=ãÉíÜçÇëW=áK aÉÜóÇê~íÉ=ak^=ìåÇÉê=î~ÅììãááK mêÉÅáéáí~íÉ=ak^=ïáíÜ=MKN=îçäìãÉ=çÑ=ëçÇáìã

~ÅÉí~íÉ=~åÇ=O=îçäìãÉë=çÑ=NMMB=ÉíÜ~åçäKt~ëÜ=ïáíÜ=N=îçäìãÉ=çÑ=TMB=ÉíÜ~åçä

oÉëìëéÉåÇ=ak^=áå=abm`JíêÉ~íÉÇ=ï~íÉê=íç=~=Ñáå~ä

ÅçåÅÉåíê~íáçå=çÑ=NM=µÖLµäpíçêÉ=~í=ÓOMø`

10% (w/v) SDS, high-qualityRNase/DNase-free

RKM=Ö=pap^ÇÇ=ok~ëÉLak~ëÉJÑêÉÉ=ï~íÉê=íç=~

Ñáå~ä=îçäìãÉ=çÑ=RM=ãäK

6× Minimal Dye Loading Buffer`çãÄáåÉ=íÜÉ=ÑçääçïáåÖ=~åÇ=ãáñ=ìåíáä=ÇáëëçäîÉÇW♦ PM=ãä=NMMB=ÖäóÅÉêçä♦ OKR=ãÖ=ÄêçãçéÜÉåçä=ÄäìÉ♦ TM=ãä=ÇeOl

Microspin Cup Wash BufferTR=ãä=NMMB=EîLîF=ÉíÜ~åçäOR=ãä=ÇeOlI=ãçäÉÅìä~ê=ÄáçäçÖó=Öê~ÇÉI=ak~ëÉJ=~åÇ

ok~ëÉJÑêÉÉ

Microspin Cup Elution BufferNM=ãj=qêáë=Ä~ëÉ^Çàìëí=ée=íç=UKR=ïáíÜ=e`ä

fÑ=íÜÉ=Éäìíáçå=ÄìÑÑÉê=áë=ã~ÇÉ=ÄóÇáäìíáåÖ=~=ÜáÖÜÉê=ãçä~êáíó=qêáë=Ä~ëÉIée=UKR=íç=~=Ñáå~ä=ãçä~êáíó=çÑ=NM=ãjIîÉêáÑó=íÜ~í=íÜÉ=ée=áë=ëíáää=UKRK=fÑ=åçíI~Çàìëí=íÜÉ=ée=íç=UKR=ìëáåÖ=ÉáíÜÉê=e`äçê=k~leK

TE Buffer (1×)NM=ãj=qêáëJe`ä=Eée=TKRF==N=ãj=baq^^ìíçÅä~îÉ


APPENDIX I

The Slide Gel Electrophoresis Technique for Analyzing Labeled cDNAqÜÉ= É~ëáÉëí= ãÉíÜçÇ= íç= éêÉé~êÉ= ~= íÜáå= ÖÉä= Ñçê= íÜáë= ~å~äóëáë= áë= íç= ìëÉ= ~R=�= TKRJÅã=Öä~ëë= ëäáÇÉI= éçëáíáçå= ~=ãáåáÖÉä= ÅçãÄ= çîÉê= áí=ïáíÜ= ÜáÖÜ= íÉåëáçåÅäáéëI=~åÇ=~ÇÇ=NM=ãä=çÑ=OB=EïLîF=ãçäíÉå=~Ö~êçëÉ=åÉ~ê=íÜÉ=ìééÉê=ÅÉåíÉê=çÑ=íÜÉëäáÇÉK=qÜÉ=ëìêÑ~ÅÉ=íÉåëáçå=çÑ=íÜÉ=ëçäìíáçå=ïáää=éêÉîÉåí=çîÉêÑäçï=~åÇ=éêçÇìÅÉ~=ëã~ääI=íÜáå=ÖÉäI=ïÜáÅÜ=Å~å=ÄÉ=ëÅ~ååÉÇ=ïáíÜçìí=ÑìêíÜÉê=ÇêóáåÖK=aç=åçí=~ääçïíÜÉ=íÉÉíÜ=çÑ=íÜÉ=ÅçãÄ=íç=çîÉêä~é=íÜÉ=ÉÇÖÉ=çÑ=íÜÉ=éä~íÉ=çê=íÜÉ=ëìêÑ~ÅÉ=íÉåëáçåã~ó= ÄÉ= ÄêçâÉåK= rëÉ= qêáëJ~ÅÉí~íÉ= ÄìÑÑÉê= áå= íÜÉ= ~Ö~êçëÉ= ÖÉä= ëçäìíáçå= ~åÇÇìêáåÖ=íÜÉ=ÉäÉÅíêçéÜçêÉëáëK=fåÅäìÇÉ=~=ak^=ëáòÉ=ëí~åÇ~êÇ=áå=~=ä~åÉ=çÑ=íÜÉ=ÖÉäIéêÉé~êÉÇ= ïáíÜ= ëí~åÇ~êÇ= ÇóÉ= äç~ÇáåÖ= ÄìÑÑÉêK= cçê= íÜÉ= ÉäÉÅíêçéÜçêÉëáëI= ìëÉëí~åÇ~êÇ=~Ö~êçëÉ=ÖÉä=í~åâëK

cçê=ÑäìçêÉëÅÉåÅÉJä~ÄÉäÉÇ=Åak^=ÇÉíÉÅíáçåI=ëí~áå=íÜÉ=ëáòÉ=ëí~åÇ~êÇ=ëÉé~ê~íÉäóáå= ÉíÜáÇáìã= ÄêçãáÇÉ= ÑçääçïáåÖ= ÉäÉÅíêçéÜçêÉëáëK= ^ÑíÉê= ëÅ~ååáåÖ= íÜÉ= ÖÉäÅçåí~áåáåÖ=íÜÉ=ÑäìçêÉëÅÉåÅÉJä~ÄÉäÉÇ=Åak^=íç=ÇÉíÉÅí= ÑäìçêÉëÅÉåÅÉI= ëí~áå= íÜÉÉåíáêÉ= ÖÉä= áå= ÉíÜáÇáìã= ÄêçãáÇÉ= íç= îÉêáÑó= ÅçãéäÉíÉ= êÉãçî~ä= çÑ= íÜÉok^=íÉãéä~íÉK

REFERENCES

NK pÅÜÉå~I=jKI=pÜ~äçåI=aKI=a~îáëI=oK=tK=~åÇ=_êçïåI=mK=lK=ENVVRF=pÅáÉåÅÉ

OTMEROPRFW=QSTÓTMK

OK _êáííÉåI=oK=gKI=dê~Ü~ãI=aK=bK=~åÇ=kÉìÑÉäÇI=_K=oK=ENVTQF=jÉíÜçÇë=båòóãçä=OVW=PSPÓQNUK

PK tÉáåÉêI=^K=jKI=aÉáåáåÖÉêI=mK=iK=~åÇ=bÑëíê~íá~ÇáëI=^K=ENVUSF=^ååì=oÉî=_áçÅÜÉã

RRW=SPNÓSNK

QK i~åÇÉÖÉåíI=gK=bKI=g~åëÉå=áå=ÇÉ=t~äI=kKI=aáêâëI=oK=tKI=_~~çI=cK=~åÇ=î~å=ÇÉê=mäçÉÖI=jK

ENVUTF=eìã=dÉåÉí=TTEQFW=PSSÓTMK

RK iÉåÖ~ìÉêI=`KI=oáÉíÜã~åI=eK=~åÇ=`êÉãÉêI=qK=ENVVMF=eìã=dÉåÉí=USENFW=NÓSK

ENDNOTES

píê~í~éêÉé®=áë=~=êÉÖáëíÉêÉÇ=íê~ÇÉã~êâ=çÑ=píê~í~ÖÉåÉ=áå=íÜÉ=råáíÉÇ=pí~íÉëK

c~áêmä~ó=~åÇ=píê~í~pÅêáéí=~êÉ=íê~ÇÉã~êâë=çÑ=píê~í~ÖÉåÉK

^äÉñ~=cäìçê∆=~åÇ=lêÉÖçå=dêÉÉå®=~êÉ=êÉÖáëíÉêÉÇ=íê~ÇÉã~êâë=çÑ=jçäÉÅìä~ê=mêçÄÉëI=fåÅK

`lqJN=ak^®=áë=~=êÉÖáëíÉêÉÇ=íê~ÇÉã~êâ=çÑ=fåîáíêçÖÉå=`çêéçê~íáçåK

^obp=áë=~=íê~ÇÉã~êâ=çÑ=jçäÉÅìä~ê=mêçÄÉëI=fåÅK

cäìçêçiáåâ=~åÇ=`v=~êÉ=íê~ÇÉã~êâë=çÑ=^ãÉêëÜ~ã=_áçëÅáÉåÅÉë=iáãáíÉÇK

instruction manual - uni-koeln.de · la jolla, ca 92037 telephone (858) 535-5400 order toll free...

Documents