INSTRUCTION MANUAL - uni-koeln.de · La Jolla, CA 92037 Telephone (858) 535-5400 Order Toll Free...
Transcript of INSTRUCTION MANUAL - uni-koeln.de · La Jolla, CA 92037 Telephone (858) 535-5400 Order Toll Free...
FairPlay™ Microarray Labeling Kit
INSTRUCTION MANUAL
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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
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FairPlay™ Microarray Labeling Kit
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
FairPlay™ Microarray Labeling Kit
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=@m^OPMMNFcäìçêçiáåâ�»=`v»=PKR=ãçåçÑìåÅíáçå~ä=ÇóÉ=RJé~Åâ=E`~í~äçÖ=@m^OPRMNFcäìçêçiáåâ»=`v»=R=ãçåçÑìåÅíáçå~ä=ÇóÉ=RJé~Åâ=E`~í~äçÖ=@m^ORMMNFcäìçêçiáåâ»=`v»=RKR=ãçåçÑìåÅíáçå~ä=ÇóÉ=RJé~Åâ=E`~í~äçÖ=@m^ORRMNF
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^obp»=^äÉñ~=cäìçê∆=PRM=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSTRF^obp»=lêÉÖçå=dêÉÉå∆=QUU=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSTQF^obp»=^äÉñ~=cäìçê∆=QUU=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSSRF^obp»=^äÉñ~=cäìçê∆=RPO=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSSSF^obp»=^äÉñ~=cäìçê∆=RQS=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSSTF^obp»=^äÉñ~=cäìçê∆=RRR=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSTTF^obp»=^äÉñ~=cäìçê∆=RSU=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSSUF^obp»=^äÉñ~=cäìçê∆=RVQ=ak^=i~ÄÉäáåÖ=háí=E`~í~äçÖ=@^JONSSVF^obp»=^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¬
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ADDITIONAL MATERIALS REQUIRED FOR HYBRIDIZATION TO
MICROARRAY
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NOTICE TO PURCHASER
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FairPlay™ Microarray Labeling Kit 3
INTRODUCTION
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4 FairPlay™ Microarray Labeling Kit
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PROTOCOL GUIDELINES
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FairPlay™ Microarray Labeling Kit 5
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6 FairPlay™ Microarray Labeling Kit
PROTOCOL
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==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
FairPlay™ Microarray Labeling Kit 7
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
8 FairPlay™ Microarray Labeling Kit
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
FairPlay™ Microarray Labeling Kit 9
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
10 FairPlay™ Microarray Labeling Kit
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
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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
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FairPlay™ Microarray Labeling Kit 11
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
12 FairPlay™ Microarray Labeling Kit
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
FairPlay™ Microarray Labeling Kit 13
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
14 FairPlay™ Microarray Labeling Kit
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
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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
FairPlay™ Microarray Labeling Kit 15
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
16 FairPlay™ Microarray Labeling Kit
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
FairPlay™ Microarray Labeling Kit 17
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
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cäìçêçiáåâ=~åÇ=`v=~êÉ=íê~ÇÉã~êâë=çÑ=^ãÉêëÜ~ã=_áçëÅáÉåÅÉë=iáãáíÉÇK