TempliPhi完全テクニカルハンドブック（PDF:583 KB）
Contents
1. L3 0000000000000 Premi 0000000 Q0000000000000000000000000000000000000000O 00000000 0 0000000000000000TempliPhig 0 00000 pod 00000000 00000000 00000000000000000000 00000000000000000000000000000000OPremxJ0000 00000000000000000000000000000000OPremx 0000 Q0000000000 Q00000000000000000000 BAC DNAQODO DO DNADO ODA Add 000000000000 00000 QDNAGO00 000000000000 000000000000000000000 Q0000000000000000000000000000000000000000 QDNAGO 000 00000000000 000000000000000000000 Q0000000000000000DNAg 00000000000 000000000 QC00000000DNADO000000 000000000000 000000000 5 1 TempliPhi DNA Amplification Kit 00000000000 0000000000000000000000000000000000000TempliPhif gd 0000000 TempliPhiD DNA 00000000000000000000000000000000 000000 DNAGO 00000 0000000000000 0 0 PicoGreen 000000 000000 O TempliPhi 100 DNA Amplification Kit 1 000000 25 6400 10 00000000000 DNAQgoD 00000000 O TempliPhi 500 DNA Amplification Kit 00000000000000000001n9090000 000000 25 6400 50 0204000000000 W0002. Go o 940180 D D TempliPhi LCH 0000000180 0 A OO TempliPhid ODNAT OO 0000000000 0000000000000000M133000000D0Q0QCforward 0000 0 1 RCA Rolling Circle Amplification 0 0 DOO DNAD OO O OO0000000 Phi29 DNA polymerase HO0000000000 O00 AQ Phi29 DNA polymerase 0000000000000 BM Phi29 DNA polymerase 0000000000000000000000000000Q000000000000000000000 000000 branchingh dd 0 00000000000000000000000DM TempliPhi 100 500 DNA Amplification Kit TempliPhi 100 500 0 0 00000000000000 25 6400 10 25 6400 50 00 01009 00 0500000 Sample buffer 0 1x 0 5 ml 5 x 0 5 ml 70 C Y O 20 C Reaction buffer 0 1x 0 5 ml 5 0 5 ml 70 0 0 20 C Enzyme mix 0 1x 20 pl 5 x 20ul 70 C Positive control DNA 1x 50 pl 5 x 50 ul 70 C Y O 20 C 2 ng ul pU 6 19 ositive control DNA 0 0 0 TempliPhi 0000000004000 0 5 ug 0 DNAD TempliPhi Large Construct DNA Amplification Kit TempliPhi LC 0 C00000000000000 0 000000 ABI 37307 310010 0000 00000000000000000
3. EJ EJ l y 4 Phi2 D A Polymerase Exonuclease 0 0000000 0 esa O DNA 4449100000 20 q 709000000000 0500001n90 000000 DNAU D 6 TempliPhi LC DNA Amplification Kit 6 N amp AAVALYARNSE GX 0169 0073 sS van ANG usa 000000000 3 25 1000000000 Red 0000000000000000 150 9001 2000K 1111 TEL 03 5331 9336 FAX 03 5331 9370 e mail Tech J P ge com Home Page http www gehealthcare co jp lifesciences 00000000000000000000000000000000000000000000000000000 00000000000000000000000000000 04 12 10 NS 0000000000000000000000000000g
4. SE 0000 000000000000 TEL 03 5331 93360 000000000000 FAX 03 5331 9370 e mail Tech JP ge com Copyright 2004 GE Healthcare GE Healthcare dd 00 000 000 0000 00 E Healthcare 00 000000000000000000 0000000000000000000000000000000 0000000 DU D 1 1 TempliPhi TempliPhi DNA Amplification Kit HB Ug TempliPhi 100 500 DNA Amplification Kit TempliPhi 100 5000 DO I BAC Fosmid DNA 000000000000 OOO TempliPhi Large Construct DNA Amplification Kit U TempliPhi LCH 00 0 0 0 0 0000 0 0 Phi29 DNA polymerase EG Rolling Circle Amplification RCA TJ 30 G 0 0 0000000000000 Phi29 DNA polymerase DOD Tag DNA polymerase 0 0 UD empliPhig 000 J TemplPhi LC BAC
5. 0 0 000000000000000000000000000000000000095 q 30 EE Co Eo EE cap ESTE 3 000000000000000000 TempliPhi Premix Reaction buffer Enzyme mix 0000000000000000 O00000000000000000000000000000000000000qQ0000 00000 Phi29 DNA polymerase 5 C 0 00000000 TempliPhi Premix 0000 000000000DNAD00000000000000000000000000000 00000000000000000000 PicoGreen dsDNA Quantitation Kit 0000 0000000000000000000000000000000000000leg 180 empliPhig 000015 ud 0 DNA 000000000000 00000000000000000000000000000000DNAgg00000 00 00 000 000000000000 0053230000 4 Q00000000000000000000000_ TempliPhid 00 00000000DNADD000000000000000000000 000000000000000000000000000000000000040 0 DOTE 8 40 10 1000 300 ngi po0000000000000 o 0 O 0 9
6. po00000001 8030pmop a 0000000090 pogo 000000000000 00000000000 0000000000000000 00000000000000 0000000 gt pH8 0 0 0 15 Q00000000000000000000000000000000000 oOo oO EDTAJ L3 x 20 80 95 0000000000000000000000000000038400000 0000000010012 0000003040 95 000000000000000 9 O Q000000000000000000 O 1 O O 8 0 3 960 0 3840 00000 5 000 romp 403300 00000000 00000000000000000000500rpm 100 000000000000000 5 0 5 00000000002500 500 uig 9600000000100 38400000000 30 0 6 D0000000000000000000s500 10 000000000000000020 5000DNApg000000000 2 7 CODO DNA DO 000000000 000000000104 00 000000000000 Q0000000000000000000000000000000000000000000 000000000050100000000000000000 2 4 DO DNAQD00000000000000000000 0384000000 Q00000000000M13000000000000000000000BACO0O00O
7. PDD 0 5 3 PicoGreen dsDNA Quantitation Kit 0 TempliPhi DNA 0000 TempiiPhi 000000000000000000000DNATd00000000000000000000000 Du Du Du D DO 0000 TempliPhid Typhoong PicoGreen DNA O Q 000000000000000000000000000000000000000000000000 DNACO 0000000000000 000000O0DOOOOPicoGreen dsDNA Quantitation Kit 0000 000 0 8 DOTyphoon 94100 00 00000000000000000 1 1 1 1111 11 1 1 1 1 1 DOLO 000000 O TempliPhi 100 DNA Amplification Kit A 000000000000 0000000 25 6400 10 1 000000100 TE O TempliPhi 500 DNA Amplification Kit buffer 500000000000 2 ng ulr O 0000000 25 6400 50 2 3 1 000 ngmg 00000002 1 Typhoon 9200 94000 0 0 0 po00000000000000000TE buffer 100 1 101 010 00 0 0 O ImageQ uant O Microsoft Excel M icrosoft B TempliPhid DN AD OO O PicoGreen dsDNA Quantitation Kit D Molecular Probes 1 TE buffeg 99 ud 0000000000000 TempliPhig 0014000000 000090000000 O 0000 NalgeN unc PolySorp 2 0000000000000 TempliPhig DNAQ O 0200000000000000 2 ng ul DNA ul TE buffer ul PicoGreen ul 000000 PicoGreeng 0 O TE buffer 200000 300 0000000000 5000 D
8. 00000000000000000000500 5000DNApg000000000 Q00000000000000000000000000000000000000000000 000000000050100000000000000000 OO Q amp A _ Im TempliPhi 100 500 00 0 0000000000 TempliPhi LC 0 0 00 A 720 9000000000 80 7 85 90000000000000M0000000a 0 0000400000000 000000000000 0000000000000000000000000000s50O 000000000000000000000000 Sample Buffer Reaction Buffer Positive Control DNAD 20 0 000000000090 A 000000 0 00000000000000000 A 80000D0000DNA 0000000000000000000000000000000 Q Phi29 DNA polymerase 00 00000 A Phi29 DNA polymerase 00 0000000000000000000 Q Phi29 DNA polymerase 0 O fidelity go AD00000000000000000000000001x10 1000000000000 D00000TaqDNA polymerase 0000000001x10 000 Q Phi29 DNA polymerase 40 0000000000000 Phi29 DNA polymeraseg 00000 Q 66 000000000000 04079 30 Co 00000000000000 ADD0000000000006GC 000000AT0000000000000000000g 0000000000000 uu Q 000000000000000000180000000000 TempiiPhig 0000000000 DNA Geno
9. 00000 00000 0 00000 0 0 00000 Reaction buffet 000000000000 Phi29 DNA polymerase OOOO 0000000000000 Enzyme mix 50 0 0000000000 Phi29 DNA polymerase 0000000 O Sample buffer 9 ug 4 55 3000000000000 Reaction buffer 9 ul Enzyme mix 0 5 u 00000000000 0000000000065 1000000000Phi29 DNA polymerase 00000000 opa 0000000000000 Oooo DO L3 c 222 Q000000000000000000000000000000000000000000000 OOOO TempliPhi LLCOOOOOOODBACOFosmid DNAGO0000000000000000 Q00000000000000000000000 1 Sample buffer Reaction buffe 400000000 2 Sample buffer 0000000090 3 Sample buffer DO 00 0 0 0 0 000000 000000000000 5 4W0 00000 Sample buffer 00000000 BAC DNA Fosmid DNA D 10 10 ng 4 ul 00
10. 19000000 qund 00000 3 0 pmol ul iu 000000 2 ul 0000000 iu BigDye 5x Dilution Buffer D DYEnamic ET dilution buffer OOO 0 ul DO 12 ul 0000000000 DO O0C 0000000000 00000000000014000rpom 1000000000000000DNA OOOO DC 0000000001 3 0pmo 000000000000000000000000000000000000 00000000000000000000000000000000 gt 4pmo go 00000M000000000 0000000000000000000000000000000000000044000084000204000 Q00000000000000000000000000000000000000000000000000000 2 00000000000000000 DYEnaic ET terminators BigDye terminators 95 20 s 95 C 20 s 50 C 30 s 20 C 5 60 C 2 min 60 C 4 min O O 60 cycles O O 60 cycles 000000000000 PCR 0080000000000 000000000 000000000000 00000000000000 000000000000000000060080000000000000025 30000 000000000000 000000000 0000000 po00000001 8030pmop a 00000000 pogo 000000000000 00000000000 0000000000000000 00000000000000 0000 00 0 00 1100 1 0000000 gt 0pH8 0 0 250 mM 15 Q0000000000000000000000000000000000000000000 Q000000 2 30 ul 96 000000000
11. 6 00000008Aco0o00000000000000000000000000000 0D 000000000000PNAQ000000000 TempliPhiLcoo00000000 Q0000000000000000000000000000000000000000 Q0000000000000000 L3 2 D D D do DO 00 L3 L3 r3 O 0 O00000000000000000000ABI3730 g C0000000000000M630000000000000000000000000000 Q0000000000 A 1 38405000005Wo 000000005000 DNA ooo ug UUDDUUDUDDNAJUUDDUDUDUUDUUDUUBACUDUDUDDNAJUDUUDUUDUDUDUUDUDUUDUUD DNAD 00000000000DNAg000000000000000000000DNAg g000000000000 2 Precipitation Mix 30 wD 0000000000000000000 Precipitation M ix 3mpog00000007mp9 p4000020mpo000000 3 5 000 rpmp 4 C 300 0 0000000000000000000000000500 0 0 Q00000000000000000 4 70 p00000003040 000000000 5 000 rpmg 4 5000000000000 00000000000000050rpm 0 000000000000000000 5 D000000000010mM Tri pH 8 008W00000000005010000 Q00000000000000000000000000000000000000000 8
12. 000000000 5 000 rpm 4 q 5000000000000 30 00000000 70 4 1000000 0 500 dodo 0000000000000 10 mM 0000 PH 8 00 10 4000000000050 ien 00 0 5 8 1 pl 10 1 111 D 3 0 pmol ul 1 ul pl 4 1 2 ul 111 BigDye 5x Dilution Buffer DYEnamic ET dilution buffer OOO 3 ul DO 20 ul 0000000000 00000000001 8030pmoi 00000000000000000000000000000000 gt 4pmol gd 0000000000000000 0000000000000000000000000000000000000044000084000204000 O00000000000000000000000000000000000000000000000000000 2 00000000000000000 DYEnaic ET terminators BigDye terminators 95 20 s 95 C 20 s 20 C 30 s 50 C 30s 60 2 min 60 C 4 min O O 60 cycles O O 60 cycles dad 000000000 PerRpap a 0080000000000 0000000000000 00000000000 00000000000 000000000000000000060080000000000000025 30000 000000000000 000000000 20000000 0
13. DNAQ O O PicoGreen dsDNA Quantitation reagent 0 0 e B 1 000 000 000000000000 000000000000000000000000000000000000000DNAGOO Q0000000000000000000000000000000000000002 1 2070 Q00000000000 0000000000000000000000000000001100 11000000000 001070 10 Q0O000000000000000000030 1 10 000 1 1 000 Yield ug 0 0 1 10 e 1 0 4 6 8 10 12 14 16 18 Time hours O DNAQ DO TempliPhi 000000 dtu 19 000000000 5 0000000 TempliPhi DNA Amplification Kit O O O 4 Q0000000000000000000000000000000020000000000 1 0 00000000000000000000000000000000000000000 000000000000000DNA000000000000000000000 2 0000000000000000 nooo DO TempiiPhi 000000000000000000000000000000000 0000000000TempliPhid 00 0000000004M000000000000000 000000000000000001U4Q00TempliPhi CO 000005 HO 0000000 000000000000000000000000000TE bufeg 000000000000 000000000000000000001uH QQTempliPhi 5 6 HOOODD mi puc TH pg al DNA added to reaction 8 2 E 55 4 puc 18 Linas p 18 Super Lol am T ammi D 4 TempliPhi DNA Amplification Kit GO DO DNAD OO TempliPhi 1 ng pUC18 DNAD 000004000120 000 000 0000 000000000000 e 3 DO0000000000000 Q
14. 223 TempliPhi LCG 0000000 DNAQ DY Enamic ET Terminator Cycle Sequencing Kit QQ ABI PRISM BigDye Terminator Cycle Sequencing Kitg DNA 0000000000000 3730 0 ABI 31000 0 00 00 00 000000000000 1 9650000000005W000DNA 5 000 0000000 DNAD 55 UD D OU UL D DO D DNA DNAD 1040 30 DO gg 0 gg 00 0000 000000000000 0000 0000000000000 90 Kb BACH ABIS730 BigDye v3 10 0000 0 0000000000000000000000 TempliPhi LCA 00000000000 BigDye v3 1 Premix 10 pl 5 pl 2 pl 1 ul 4 550 bp 670 bp 5 bp ul 325 bp 650 bp 603 bp 503 bp 2 pl 113 bp 230 bp 491 bp 2 bp 2000000 1 0000 mlr 7 95 3M Precipitation 2 Wi0 D0000000000000000000O 30 rpmp 4 41300 00000000000000000000000000500 rpm 0 5 000 3
15. DOO a rici 20 00050 2 00000000000000000 DYEnaic ET terminators BigDye terminators 95 20 5 95 20 5 20 C 30 s 20 C 30s 60 1 2 min 60 C 4 min O O 60 cycles O O 60 cycles silvi AOL 0000000 1 2 3 4 5 7 8 po00000001 8030pmop a 000000000 DODO 0000000000 0000000 00000000000000000000000 Q0000000000 Q000000000000 000000000000000024 10 lt 0000000 15 8 05 0 DTADODEDTAD OO 00O000000000000000000000000000000000000000 00000 oOo z0 4 El 6 0 m0 80 95 00000000000000000000000000000000000 EDTA O000095 Q000000000000000 960 0 D 0 5 000 remp 30000000000 O00000000000000000000500 romp 19 00000000000000000 Q00000000000000000000000000000000000000000000 0 5 MO 0000000000250 500 g 600000000100 70 e
16. Ea E Oo El DOOD TempliPhi 0 000 3 pl 0000000078 pH8 500 00 Primer 3 2 pmole v 0000000 BigDye Terminator Cycle E 1 Sample buffer 5 yig O O O Sequencing Kit vera 00000000 95 CO 30000000 w Reaction Buffer 5 ul g Enzyme mix 0 2 ul 000000 000000000000 96 C 10 sec 5 0 0 50 C 5 sec 250000 v 60 C 4 min 30 0 6900000 65 1000 4 9C soak a a SE 000 ABI PRISM 31000 DODO 0 ABI Model 37300000 eS cen 0 0000000 DO0000000000000 0 0000000 000000 8 88808 00 500000 0000000000 nesteddeletion 0000 100000000 6 MegaBACE 10000 D 4000 DYEnamic ET Terminator C ycle Sequencing Kit for MegaBACE TempliPhig OOO TempliPhir 00000 00000000 TempliPhi 10000 DNA Amplification Kit TE T e Nea phina 0000 0 000 00 000
17. 0000000 0 600000 0000000000000000000 0Q00000 000 U E 00000000000100000 TempliPhi 0 000 1 yl 000000000000 Milliporen Primer 2 5 pmole Multicreeng 900000000000 Denature buffer 10 ul 0000000 DYEnamic ET Terminator 000000000 Cycle Sequencing Kit for MegaBA C E 95030000000 v TempliPhi PreMix 10 uig 95 C 10 sec 50 18000000950 50 0 95 C 25 sec 30 C ios 300000 40 C 2 min 60 C 10 sec 95 C 1 200000 60 C soak 4 HERE Bal 00000000000 0000000040060 95 0000000000000000000 00000000 00000 0000000000000 Q0000000000 00 00000 0 00 200000000000 000000001100 18002100000 odo 0000000 000000000000 0000000000 TempliPhi LC Rolling Circle Amplification Kit 00000000 0 51 x E El E El E El 40 m 0100000000000000000 1 20 1 500 0 00 4410 D DNAD 00000 m pri 5 Q Oa 5 Q 02 0000000000000 03 1 3 EY Bo C3 C3 ZOO Zi P B E EV ESI ESY E53 rod 32 D Dn ESI 3 EY Ear am Ono 6 Or
18. m DNA Hawkins T L et al Whole genome amplification applications and advances Current O pinion in Biotechnology 2002 13 65 67 Wa l Ag 8101888900 0000000 nn empliPhi DNA Amp il ication Kit TempliPhi DNA Amplification 000000000000000000000 09 00090000000000000000 0 0 0 Sequencing Circles Q00000000000000000000000000000000 34 00 00 00 00000000 00000000 0000000000 0000000 ii Plasmid CONA QO00000000000 ee eor a eR C e 35 00000000000 000 spo00000000000000000000000000000000000 36 000 0000000 00000 0 00000000 000 0000000 ia C eae PE C 37 0000 00000000000 00000000000000 000000000000 MH HEIN ee err 38 000 00000000000000000 0000000 000000000000000 1 0 0 0 00 0000 nested deletiong 0000000000000 39 00 00 00000 00000000 000000 00000 0000000000000 DO0000000000000 nnonnunnunnnnnbNADDDDUDDD f 000 0000000090 5 000000000000 ABI PRISM 373 Thermo Sequenase Il Dye Terminator Cy
19. 000000 0 00000000000000000000000000000000 A TempliPhi LCH 00 00000000000 BAC 0 00000000 000 Dg pg gg gg 0 0000 0000000000000 00000000 000000000 Q 0 0 0000080008000 0 0 000 0 0000000 AD000000000000000000000000000000000000000O0DNA 0000000000000 DYEnamic ET Terminator ABG 000000 BigDye terminator ver 3 10 Applied Biosystems p 0000000000000000 Applied Biosystems DNA sequencer 00 00000000000000000 9 00000000000000000000000000000000000g A 000000000000000000000000000DNA0000000000000a rrr rrr 0000003pmolg nn Co D000000000000BAC000000000000000000000000000 O00000000000000000000000000000000000000000 2040 000000010 0 0000000000020 4000000000000 e 00 01 00 000000000000 000 0001W00000000 660000000000000000000000000000000 000000000800000000000000TempliPhi 100 5000 0000000DNAD 01 5 uga 0000000000000 TOTO 00000 00 000000 000 000 TempliPhi 100 5000 000000000040000000000029D TempliPhi 00 DD D D D 1800000000 DNAD DO Sug 0000010000 2 50 2 25 2 00 1 75 1 50 1 00 0 75 0 50 0 25 DNA Yield ug fo a 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Time hours 0 2 TempliPhi 100 DNA Amplification Kit 000 1 ngo pUC19 DNA 0000240000000 00000000000 Triplicateg 4000000
20. Oyra GE Healthcare TempliPhi DNA Amplification Kit TempliPhi DNA Amplification Kit 111 1 1 11 1 1 1 O00 DNAg OOO 000000000000 ver 2 TempliPhi Rolling Circle Amplification Kit 00000000 nodan TempliPhi DNA Amplification Kit 11 1 ver 2 TempliPhi 100 DNA Amplification Kit TempliPhi 500 DNA Amplification Kit TempliPhi Large Construct DNA Amplification Kit TempliPh 000000000 GE Healthcare TempliPhip 000000000000000000000000000000000000000000000000 Q00000000000000000000000000000000000000000000000000000 Q00000000000000000000000000000000000000000000000000000 Q00000000000000000000000 Q0000000000000000000000000000000000 00000000000 FAX 03 5331 9370 po00000000 00000 0 000 O 0000800 0000000000000000000000000000000 uuuuu GE imagination at work 9 Contents eee Li MII en 5 TX EMA id irm rem tu tendu
21. Phi29 DNA polymerase 00 0000000000000000000000000000000000000000 00000000000000000 Phi29 DNA polymerase 00000000000000000DNAgG00000 O00000000000000000000000000000000000000000000000000000 0000000000 Phi29 DNA polymerase 0000000000000 9 DODNACO 00 000000000000000000000000000000000000 000000000000000000000DNAd 0000000000000 000000 0000000 Q0000AQ 000000010 20 1509 300 n0 000 00000000000002040 0000 00000B0 buffer 20 30 0 000020 10H 2040 0000000000000 TempliPhi 0 0 OQ 10W0 0000 1 250 1 75 ug DNA 0000000000000 20 900000 0000000000 TemplPhi 0 DNA JI D Q0000000000000000000000000 0 2 1 3 Control DNA 000000000000 po00000000000000DNADO 19 0000 DNAD 2 50 000000 1 Control DNA 0 5 ul 1 ng O Sample buffer 5140000000 2 900002120 4000000000 3 D00000000040000 59 00 DNA 0000000 2 2 TempliPhi Large Construct DNA Amplification Kit 0 TempliPhi LCO 0 0 0 2 2 1 00 00000 Sample buffer Reaction buffer Enzyme mix 309 0000000000000000 Sample 0000000000000000 paN 0000000000000000000 00 0
22. Primer 3 3 pmole 3840 70 00 000000000090 1 00000000000 100000 v Denature buffer 10 ult O O 9550 30000000 0000000 BigDye Terminator Cycle 00 000000000000000 Sequencing 000000000000 v 000000 TempliPhi PreMix 10 uig 0000000000 000000 v 96 2C 10 sec 30 180 00000 95 cr 500 50 9C 5 sec 250000 60 C 4 min 4 C soak T D Eo p 4 ET maar E Em ESTE 6 ene gt TempliPhid O O dodo 0 00000 lx ia Lawl Apes venire bee ica TempliPh 000000000 800 0 800 0000000 000000 O C elegans DO 8 000 U O000000000000000000000000000000 O00000000000000000000000 000000 O00000000000000 5000000000000000 00000000000 e 0 0000000000 ABI PRISM 3100 373 BigD ye Terminator C ycle Sequencing Kit ver 3 TempliPhi 0 O TempliPhig 0 0 0 0 GeneAmp 9700 TempliPhi 100 DNA Amplification Kit f B B AA AE Eo 000000000000000000000000000000000000 00000000PCR0D0000000000000000000 E E
23. 6000 00 PicoGreen dsDNA Quantitation Kit 0 DNAD DO D DO 96 D D 00 D u D DO poeo000080000000 1 000 ng mig DO CODOR 0 9960 0 0000000000 000000000000000000 00 Q000000000000000 Q00000000000000000000 0000000000 TempliPhig DNAD I Q0000000000000000000000 Q000000000 6 UD E TempliPhi Reagin MJ Giesler TL Merla AL Resetar Gerke JM Kapolka KM M amone JA TempliPhi A sequencing template preparation procedure that eliminates overnight cultures and DNA purification J Biomol Tech 2003 14 2 143 8 E TempliPhi Nelson JR Cai Y C Giesler TL Farchaus JW Sundaram ST Ortiz Rivera M Hosta LP Hewitt PL M amone JA Palaniappan C Fuller CW TempliPhi phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing Biotechniques 2002 Jun Suppl 44 7 E TempliPhi begomovirus DNA Inoue N agata AK Albuquerque LC Rocha WB Nagata T A simple method for cloning the complete begomovirus genome using the bacteriophage phi29 DNA polymerase J Virol M ethods 2004 116 2 209 11
24. N O 00000000 O O Sample buffer 5 uy 1 uf 9 300000000000000 OOOO Enzyme mix Reaction buffer OO 30 40180 000000000000 0000000000000 esq 10000000000 Phi29 DNA polymerase 00000000 00 000000000000000000000000000 0 1015 0 0 DNA 2 1 2 TempliPhi 100 5001 0 0 OOO Q000000000000000000000000000000000000000000000 00000000000TempliPhi g go D00000000DNAgC OO 0000000000000 000000000000001015ugdAdDNAGOC0 000 000000000000000 O O O O Sample buffe Reaction buffer Enzyme mix 0 Positive control DNAD 0 OOOO xp 00000000000000012000000000 1 Sample buffer Reaction buffe 400000000 2 Sample buffer 5 ul 0000000000 3 Samplebuffet D0 00 000000000000000000000 000000000 00000000 1 7000 1 109 0 0 100 10 ceisd0 0000000000000 00 50 p0 TE buffer 0000000000000000 0 0 20 0 5 ur Sample buffer 000000 00000 000000000 or 05 O O Sample buffer 000000 00000000000 0000000000010 00 0 50 TE buffet pH7 5 8 00 0000000 00000000000000 0 0 0 20 0 5 ll Sample buffer 000000 gt 00000M139000000 ul D Sample buffer 00000 0 5 0 20 0000000 00000 000000000000 Sample buffer 00000
25. TempliPhig 0000 O TempliPhi 10 000 DNA Amplification K it 00000000000000000000000000 0 0 0 25 6400 01 00005000000000000000000000 DODO 000000 0000000000 000 1 1 0 EcoRI 0 0 0000 000000000000090 00000000000000000TE buffe 00000000000000000001 0000 TE buffer 1 O TempliPhi 10 000 DNA Amplification Kit 25 6400 01 O TempliPhi 100 500 DNA Amplification Kit 0O 0000000000000000 sud 000000 sterile LB media 1xLB standing culture OD 0 7 0 5x LB shaking culture OD 1 22 1X LB shaking culture OD 3 Product ug RCAQ Added Media ul OOOOO000000 TempliPhig 0 000 020000000001 ng pUC TempliPhig 0 00 00 TempliPhi 10 000 DNA Amplification Kit 00000000000 PUC 18 pg o S 250 62 15 lt 0000000 pUC 18 linear 0 5 10 15 20 25 30 Time hours 06 DNA 000000000000 07000000000000 TempliPhi 10 000 DNA Amplification Kit 00 1 ng pUC18 DNAD O O TempiiPhi 0000000012000 2 D
26. 0 00000000000000000000000000000000000 TempliPhiQ D000000000000000000000000000000000 00000000000000000000000000000000000000LB 00000000000000000000000000000000000000000000000D000TE buffer Pe ee ae ee eee J C00000000000000000000000 000000000000001 1000 1400 D 0 100 10 cells 1101000000 0 0 50 Q0D0TEbufe 0000000200 5u000000000000000000000000000000000 TempliPhiQ 1110 0 0 0 0 105 cellsg00000000000000000000000000 gt 00 000000000000 DNAD 000 1 10 DNAQ 0 5 ul O O Sample buffer 1 1 DL 4 O0000000Sample 0 000000950930 000000000000090 000440 0 00 0000 d du ooo E oom 5 TempliPhig Premix O Reaction buffer Enzyme mix 00 0000000000001000 O O 5 Reaction buffer 0 2 ui Enzyme mix 0000000000000 Premixg OO Sample buffe 00000000000000000 C000000Premx 000000000010000000000000000000000 mizi 0 00000000000000000000000 DIE OOOO 0000 1 6 500000 Premixo 0 540 4000000 Sample buffer OO0000 000000000000 000000001800 000 07 180 40 7 00000000 00000000000000000000000400000012501 75ug0 DNAGO DD D LO D 4000 1 250 1 7519 00 0000000000000040000184000000000000000000000000000 8 400000654 100 000000000000000 00000000000
27. 0 000000000029031000000000000Q TempiiPhi 0000000000 00 00000000000000000 000 0 PicoGreen dsDNA Quantitation Kiti M olecular Probes DO 0000000000 OOOOOOOOOOOOOOOO 000000000000NA 000000000000 169 180 0 O TempliPhi 000015 ug OO 1 1 5 TempliPhi Premixg Reaction buffer Enzyme mixQ 00000000000000240000000000 00000000000 0000000000 TempliPhi Premix9 00000000000000000 000000000000 Phi29 DNA polymerase 0 8 TempliPhig DNAD OO DO0C 00 000000000000 0000000000 0000000 8 uuuuuppunuunupupuupupupuupun TempliPhig 40 ug TE buffer O O 090000 2 e El Q0000000 9 D0000000000000000000000 TempliPhid 0000000 0000000040 10 ur 1000 300 00000 L
28. 000 A TempliPhi DNAQ branched concatemerg 5000 0100000000000000 0000000000000000000000000002000000000000000 000000000000000000TEpuffe ooo000000010 2000000000 Q TempliPhig 00000000000000000000000000000000 O0000000000000000000000000000000000000000000 O PicoGreen dsDNA Quantitation 11 1 1 0 000000080 Q TempliPhid 0 00000000000000000000000000000000000 A0000000000000000000000000000000000000000000 000000000000 amin 100 5000 0 0 0 Co E 0 000000000000000000000000000 A0000000000000000000000000000000000000000000 000000000000000000000000107100 0000000001 41000 111 00000000000 000000000000 0000000 000000000 DODO 0000000 0000000000000 0 00000000000000000000000000000000 ADD1mmO00000000001 100 1 1000010710 celsg 000000000 0000000 100100400000000000001H00000000000000000 000000000000000000000000000 x 6 0000000010D nmgggg 0 looo 00 00000 Cc 00 0 0 0 0 0 0 0 00 00 0 0 00 A0000000000000000000000000000000000000000002 0000000000000000000000000000000000000000000 00000000000000 0 000000000000000000000 A 000000000000000000000000000000000000000000g 00000000000001800000
29. 0000 TempliPhi 0 0 00619 DNAQ DD O TempliPhi 100 DNA Amplification Kit A TempliPhg 0000000000 000000 25 6400 10 1 Sample Buffer 5 pi 1 ng pUC 19 D O TempliPhi 500 DNA Amplification Kit 000000 25 6400 500 2 953 300000000000000000 EcoRI 3 5 Iig Reaction buffer 0 2 ulg Enzyme mix D d TempliPhit 0000000 O TA DNA Ligase Note O LB Broth 0000000000000000000 8 0 Enzyme mix OO 0000000000000 O LB Agar 4 300180 90000000 5 65410 0000000000000000 000000 t 6 90W0 5 00 000000000090 O ElectroM AX 56 E Cells Invitrogen 7 DO 000000000 O Disposable M icro Electroporation Chambers 8 0000000000100 000000000000 O Invitrogen 9 B O Cell Porator E coli D00000 000008 000000 Invitrogen O Subcloning Efficiency DH 5a Competent Cells O Invitrogen O PicoGreen dsDNA Quantitation Kit M olecular Probes 2 3 2 uy 4 5 1 1 Q00000000000000000000000 000000 TempliPhif D00000000000000000 OO pUci9 DNAOOOOOOOODRHSAOOOO Q00000000000000000000000 Q000000000000000 000000000LtB00037Q90000000 0000000000000000000000 30 0000 TempliPhi 000000000000000 000 O O pUC19 DNAD 000000 EcoRI D 37 200000000000 20 units EcoRI 2 ul 10x buffer 20 pln
30. 00000000 Co Q 6 A BACHO 6 0 0000000000DNAD0000000000000000 A 49 17 D000000000000000000000000000000000O0DNAgO 00000000000000000000000000000000000180000000 0000000000000000000000000000000000 9 549 000000000000000000000g A 0000000400000000000 TempliPhi LC 0 O00 Q D Co O TempliPhiLCO0 0000000000000000 0 000000000000000000 A 0000025 0000030q00000000000000PNA0000000000 A 0000000301D00 Co 018000000000 000000000000000 00000000 DU 000000000000000000000 soco 00000000 un A U DO DO 0 000000000000000000000000000 OO Uu 0000000200050000000000000000 000 A 100000150 Vi 0 0 0 00 0000 000 000000002090 1 0 p0000000Votex0 0000000 4 9 Sample Buffer 000000000000 0000000000000 Q 10 00 000 0000000000 A gg00800000000000000000000000000000BAC DNA 10 ng D 0000000204900 0001m000000000000005000000250 ug 000000000 Sample Buffer Reaction Buffer 0 0 450 pip Enzyme Mixg 25 ul
31. 00000000000 00000000000000 00000000000000 25 6400 80 Dann 01000000 Sample buffer 0 1x 9ml 70 C 0 20 C Reaction buffer D 1x 9ml 70 C 0 0 20 C Enzyme mix 0 1x 05 ml 70 C s0000000000000000000000000 RNase A TEG buffer BAC DNA 000000 25 mM Tris HCl pH8 0 10 mM EDTA 50 mM glucose 100 ug ml RNaseA Lysis solution BAC DNAD 1 B U U 0 2 N NaOH 1 Yo SDS 3 00 8 2949 0000000114 mp 0000001000 0000 09 000000BACDNAJODODDO D000000000BACDNADODDOoDO TE buffer 10 mM Tris 1 mM EDTA pH 8 00 0 0000000 TempliPhi 100 500 O O TempliPhi LcH 0 0000 1 3 000000000000 000000000 http www gehealthcare co jp lifesciences TempliPhi 8000000000000 14 000000000 0000000 TempliPhi 100 500 DNA Amplification Kit TempliPhi 100 5000 00000 0000000 TempliPhi Large Construct DNA Amplification Kit TempliPhi LCO O O 2 1 TempliPhi 100 500 DNA Amplification Kit uy TempliPhi 100 5000 0 0 2 1 1 00 00000 Sample buffer Reaction buffer Enzyme mix 3r 0000000 control DN Ap D O O O Sample buffer 000 000000000000pdN JA 000000000000000 0000000000000000000000DNADD00000000000ODNADOGODO 00000000 Reaction buffer 0000000000000 Phi29 DNA polymerase J 0000000000000 0O0qgEnzyme mix 500 0000000000 Phi29 DNA polymerase pda
32. 0000000000000000000 Ears TempliPhi 0 0 00 2 000000000000 AutoSeg G 100000000 Primer 3 3 pmole 500 0000000000000000 0000000 BigDye Terminator Cycle 00000 Sample Buffer 5 WD OOO Sequencing Kt 00 000000000 95 0 30000000 0000000 v Reaction Buffer 5 uig Enzyme mix 0 2 ul 000000 0000000000005 u 96 C 10 sec 0000000000 50 C 5 sec 250000 v 60 C 4 min 30 4000000 65 1000 4 9C soak le Pla Ba me com w 0000000000000000000000000000000 Mh Sire 0000000000000000000000000000000 Tea sei esse 1000000000000000000000000000000 TempliPhig 00000000000 u 800 OOO 8080000 u 000 0000 00 00000 00 00 000000 0 O00000000000000000000000000000 000000000 00000000 0000000000000 000 0 u u 0 u 280000000000 Tes 00000000000 000000000000 ABI PRISM 3700 BigD ye Terminator C ycle Sequencing Kit ver 3 TempliPhig O O O TempliPhip 0 0000 GeneA mp 97009 3840 0000000 TempliPhi 10000 DN A Amplification Kit Rape Re Rls PR Em0 m0 O 6 o 000000000 00000000000000000000000 mnm TempiPhnnnn 140 dH200 120000 000000000000 Whatmanng
33. 0000000000000000000000000000 EDTA O000095 Q000000000000000 3 384000000 5 000 rpm 4 30000000000 D0000000000000000000500 rpm 10 00000000000000000 Q00000000000000000000000000000000000000000000 5 30410 70 000000DNAgap 00000000 6 00000000000000000000s500 rem 1 000000000000000020 2 7 CODO DNA DO 000000000 000000000104 00 000000000000 Q0000000000000000000000000000000000000000000 000000000050100000000000000000 225 000000000000000000000 TempliPhi CO O00 0000000 DNAQ DYEnamic ET Terminator Cycle Sequencing Kit 0 0 ABI PRISM BigDye Terminator Cycle Sequencing 000090090000 00000000000ABI 31009 000 000000000000000 Mis 0000000000000 0000000000000000000000 384 0000000000000224000000000 1 000000 0000 10 pl 20 00000 3 0 pmol ul 10 0 0 4 ul 0000000 2 ul BigDye 5x Dilution Buffer DYEnamic ET dilution buffer OOO 3 ul DO 20 ul 6 10 30 DODOODDNAD do T00000000000000DNAg 0000000000000
34. 0000000000000000000000000000000000000000000 Q00000000000000000000000000000000095 9 30000000 000000009509 0000000000000000DNATOOOOCORCATDODO Q0000000000000000 6 a 0000000000 Phi29 DNA polymerase 0 0 70 Q 000 80 0000000000 200 0 000000 000020000000000000000000000000000000000000000 0 00000000000000000000000DNAgG000000000 4 5 DNAJODODODO 1 011 0 0 98 0 00000000 00000000000 00 0 1090000000 Q00000000000000000000000000000000000000000000 mi 6 DNA 0000000000 Phi29 DNA polymerase 00000000000000000000DNADD000000 0002 0000000000000000000000 e 1 000000 0 B 80000000000000000 8 0000000000001 0000000 06 0 QC00000000000000000000000000 2 0000000000000003000000000000 Phi29 DNA polymerase 000000000000000000 DNATO00000000000000000000000000000000000 D0000Phi29 DNA polymerase DO
35. 3 L3 TempliPhid 0 0 0 0 000 1 Sample buffer 5 ul 0000000000 2 Sample buffer D0000000000000000 3 4 5 7 Q0000000 pa0d00 0000000000 0000005040 TE buffep po 0000000 pad 0 000 00000 0000000000 2 ulti 0000000 Sample buffer 00000 0000000000 Sample 000000 1009 1100 00000000 107 1000000 00000000 0 1 e in 0 1 11110 0000000 Samplebuffet 0000000000 p0020054p000000000 O0000M1397000000000 000000 Sample 0000000000 020054000000 1 O epo000000000000000 1000000000000 50 TE buffer O O 0000000000000 0 0 Sample buffer 0 0 0000000000000 020054000000 e000000000000000NA M13DNAD ODO 0000000 Sample buffer 1 9 10 ngi 05 1 000101000 0000 00000000 Sample buffer 0000000 95 C 30 00000000000004qQ000 00000 TempliPhi Premix Reaction buffer 5 ul Enzyme mix 0 2 id 00 000000000090 0000 Premixg 5 uig Sample buffer uu 30 C 40180 000000000000 000000 65 q 1000000000000000 00000000 000000000000000000 5 2 TempliPhi DNA Amplification Kit 0 0 00 00000 DNADO000O000O D TempliPhi DNA Amplification Kit OOOOOOOOODNAOOOOOOOOOOOOOOOOOOOOOO Q000000000000000000000000000000000000000000000000000O DNATO000000000000000000000000000000 000000 Templiphi000000000000000000000000000000000 0000000000000 8 0 PicoGreen Q0
36. 65 2000000000000 0000000500000 3 1074 DNA Ligase buffer 1 ul T4 DNA Ligase 6 units 0000 16900000000 30 0000000000000DHs5agd0000 Q00000000000000000000000 Q0000000000000 DO 000000000tB0003 000000000 30 0000000000000000000000 DODH9DNAD 000000 OU pUC19 DNA 1 ngi TempliPhi DNA Amplification Kit OU U D D do 0000000TempliPhig O O O ID Enzyme mix TempliPhi 0 00 0000000000000 00000 puUci9 DNAD E coli DH5a 00000 O00000000000000000000000 O0000000000000000 ooo 1 go Q00000000000000000001 0000000000000000900 pi DO o O Oooo iPhi CT 00 000000000000 TempliPhig DO puCc199 n 0 000000 OOOOOOOOOOOOOOOO Oooo pUci9 DNA 9000000000 1 1 000 0000000000 TempliPhig 0 0 000 000000000000 Q00000000000000000000000 TempliP hig 000000000 00000 0000000000000000TempliPhig O 000000 ug DNAD triplicate 0 O 000000000000
37. 8 U TempliPhiQ 0 0 0 0000 go po000 000000000 O0000000000000000 1 20 1500 00000000004 celisg go 0000000000000000020u0 0 10 10 000 1105 1450 0000000000000 DOTE buffeQ 000000440 00000000000000 4 000000000090 Sample buffer p0000009 300000000000000D0 0000000000 00000 0004 0000 0000000 3 mun Ven MEE 5 TempliPhi Premix O Reaction buffer EnzymemixG 010 0000000000001000 009 pig Reaction buffer 0 5 ul Enzyme mixp Premix O O Sample buffer 00 00 0000000000000 Q000000000000Premix 00 0000000010000000000000000000000 Premixo 000000000000000000000000 000000000 40000000 95410 Premixg 00500000 6 000000000000000 180 30 7 0000000 8 654 10p000000000000000000 00000000000 Phi29 DNA polymerase 000000000000000000000000000000000000000000 00000000000 Phi29 DNA polymerase 0 010 0000000000000DNA90 000000 9 DODNATO 000 00000000000000000000000
38. NA 00 000000000NAg 00000 TempliPhi PremixD O 0000 TempliPhid 00040000000000000000 6 0000000000 SEPNAOTOOTempiPhig 000000000000 000000000000000000000 00000 22000000000 000 00000000000 DOD TODO UNU UNANINI D0000000000000000000000000 D0000000000000000000000000 D0000000000000000000000000 D0000000000000000000000000 TempliPhig 0 00 00 00 0 DNAD DO D0000000000000000000000000 3 000000000000000 00000000000000000000000000 00000000000000000000000000 00000000000000 0 00000000 000000000000000000000008 Q0000000000000DNAG000000 uuu OOOTempliPhiQ 0 0 0 0 00000 0000 0 0 4 TempliPhig 0 0 00 000000000000 O OOOOOOOOOOOOOOO Phi29 DNA polymerase 0 0000 70 0000 1 80 09000000200000000000000 20 000000000000000000000
39. NAD ng ml 1000 Lo E 5 95 100 NS 98 100 100 0 100 100 C Typhoon 0000000000000 D 00000000 1 Typhoong n n D D 1 Gray Color Adjust 010 0000000000000 0000 2 PicoGreng 0000000000000000 0 Excitation 2 000000000000000000000000 ad am 000000000000000000000 532 nm Typhoon 92000 0 0 M 3 D0000 00000000 000000000000 Emissiong 0 O O 4 Microsoft Exc d0000000000000000 520BP Typh 9400 000000000000000000000000 0 080 0 0000000000000800 PMTO OOO 0000000000000 TempliPhi DNAD O 0000000000000000000000 0000000 000004000 9000000000000 mr 0000000 D D Ve o ea OOOOOOOOOOOOOOOOOOO DO0000000000000000 1 000 9 500 0 0 nomig 200 ng mig 100 ng mi 50 ng mi 20 ng mi 10 Dun ng ml 0 ngmln 10p ImageQuant U Excel 0 0 0 0 0000000000 000000110 30 60 0000090000000 0000000000 DNA O O O ng mig 1 000 500 200 100 50 20 10 0 090000000000000 Typhoon 94100 0 0 PicoGreen 0000 20 15 10 81 5 _ 0250 500 750 1000 0 1 250 1 500 1 750 E 300 DNAD R 0 9964 00000 100000 x10 D 11 TempliPhig 0000000000 010 0 00000000 3
40. cle Sequencing Kit TempliPhi O TempliPhir 0 0 0 0 0 PCRO D TempliPhi 100 DNA Amplification Kit O GeneAmp 96000 00000000000000 ANA a 000000000000 0000000 PCR0O0000000010 0 00000 000000000000000000000000 0000 TempliPhi 0 000 2 yl 0000000000000 Primer 5 pmole 00000000000000003H h 0000000 Thermo Sequenase 0000000000000003 ul Sample Buffer 5 ul n O n Dye Terminator Cycle Sequencing Kit 000000 95 Co 30000000 w Reaction Buffer 5 ulg Enzyme mix 00000 02 9000000000005 95 C 20 50 C 15 sec 300000 60 C 1 min 30 1800000065 C01000 4 soak T o o a oo g TempliPhid 0000000000000000 00000000 Thermo Sequenase II 700000000 600 bo 0000000 DO00000000000000 0 0 0 DABI PRISM 37390000 ALF express ALF express IN 00000000000 ere l la iere OO0000 40000000000 0 00 000000 Q000000000 Q000000dA 0000000 08080005 8 0 8 800 880 0 0 0 80 0 Plasmid cDNA 0000000000000 5 0000000000 eppo 000000000 DYEnamic ET Terminator Cycle Sequencing Kit RISA 3840 000 Tem
41. miPhi DNA Amplification Kit 25 6600 019 000000000 Q 0 0009 00 00000000 uu A ODODNADDODNAJDOO 000000000000000Templihig go 0000000 GenomiPhi DNA Amplification Kif 25 6600 019 000000000 Q 00000000 95 300000000000000 Q 00000000 3 9 Q 65 C 00 0 00 00 0 00 00 000 00 000090 A Phi29 DNA polymeraseg 00 00000000000000000000000000000 000000000000 000000000000 000000000000 0 A0000000000000000000000000000000000000000000 PicoGreen dsDNA Quantitation 0 0000000000000000000029 0310000000 Q DODODNATOUVO 000000000000 A popo0000090000 0000000000000 00 000000000000 0000 PicoGreen ds DNA Quantitation Kit Molecular Prrobes 000000000000 0 A 000000000000 65 100 00 000000000000 00000000000 0000000Phi29 DNA polymerase 1000 0 000 000000000000 2209 0000 040000000000 Q 00
42. pliPhig TempliPhir 00000 TempliPhi 10000 DNA Amplification Kit 11 1 0 0 w T ran EE a a 11 O 000000000000000000000000000000000 00000000000000000000000000000 68 000000000000000000000a000 00000dH 00 1 5000 TempliPhi 0001200000000 000000000000 Milliporeg Y 2 000 Primer 3 pmoleg B DB D Multiscreeng 90000000 D enature buffer 10 uig O O DYEnamic ET Terminator Sequencing Kit 0000000000000 95 C0 30000000 v TempliPhi PreMix 10 uig O00000000000000 956 10 C 10 sec 600000 50 5 1600000095 C0500 60 C 1 min 30 42C soak T E ORI ERE li 2 wi AMI insedit ls 1 as als Rom ls ata d 11 IP haad AGO E TAV 4 1 A Ya M in T 2 z mu TRIN ATA Ed P Fl U D OO 0000000000 0 0 0000005 0080000000000 00000000 000008 go00080 00000000 000000000000 0000000 2000000 i aa ee 6800 000000000000 ABI PRISM 310 BigD ye Terminator C ycle Sequencing Kit TempliPhi 0 O O TempliPhig 0 0 000 PCRO DO GeneAmp 24000 TempliPhi 100 DN A Amplification Kit ggmmBBBBBaaggu O 000000000000000000000000000000000 0000000000000000
43. rends 5 ek mn FA Fe rm 5 di AAN A a su a sa aa codicc nr 6 LA Ep LISTER EE eerie eite et e lt dnte 6 r RR wa ka AAA TE s 7 2 1 TempliPhi 100 500 DNA Amplification Kit O TempliPhi 100 5000 Aa 7 2 0 CETTE TONO eC IT DIS TETTO 7 2 1 2 TempliPhi 100 5000 DOO OODNAQ OOD 7 213 0 09 8 000000000000 3 53 35 9 9 2 2 TempliPhi Large Construct DNA Amplification Kit H FemipliPhi LCT 9 221 Id Betis PA DUCK NAA EA Rode a E FER DER a ete ated o dune 9 222 0 E DNADM I Eli nac teen crie et f nei dete e ol Rh 9 223 eee n n n M n 11 224 QODNAD000000000000000000000384000000 13 225 000000000000000000000 eee e 15 3 J J Q amp A uo PROP EMO 17 42 111311 I 10111 11111 19 5 0000000000 24 5 1 TempliPhi 0 0 0 0 0 BLU OO ODD aa 24 5 2 TempliPhi tt 0 0000 000 0DNADO DODO El D bc 27 5 3 PicoGreen dsDNA Quantitation Kit 0 00 TempliPhir O DNAD O ia 29 cilea 32 000 TempiiPhi D 000000000000000000000000000000 0000000000 Co O000000000000000000000000000000 0000000000000000000000000000000 O000000000000000000000000000000 000000000000000000000000000000 00000000000000000000000
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