I はじめに - 同仁化学研究所
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1. 00000000000000000000000000 000000000000000000000 40 00 PBST 000000 Washing Buffer 0000 1 EL 00000000000000000000000000 0000PBSTD 00000000000 0000000 5 DNAJ O 000000 0000000000000000 0000000DPNa00000000000000000 0000000000000000 6 ARPO 0 0 0 DB 0 D Filtration Tube 000000000 0000ARPO0000000000000000000 00000000000000 7 170000 10 O Filtration Tube 0000000 EQ DNAT 1 Hl D CO D I D B D D 7E D D 1 Filtration Tube 0 0 0 0 0 0 DNAD O O TE Buffer D O 000000 20000000 DNA0000000000000 lt 000 gt Dojindo Molecular Technologies Inc 211 Perry Parkway Suite 5 Gaithersburg MD 20877 Telephone 1 301 987 2667 Fax 1 301 987 2687 URL http www dojindo com lt 00000 gt poOdgodo0000 Tel 096 286 1515 0 00 D 0000000 0 0 Tel 03 3578 96510 0 Fax 096 286 1525 Fax 03 3578 9650 General Protocol at a Glance Day 1 overnight General Protocol OO LI L LU L U U U UI U U U LL U UU UI U LU 1011 27 OO DNAD O O 10 uL 9 ARP Solution O00 37 cp 00000000000 a0 0 00 SNAG OO 0eLDOO000000000 ca ARP Solution 0D O O Y cup 5 Step1 D D 0 C ARD O Lj spin ARPOO DNA LI Tit UU ARO oOo T 400 pL N 201 DO 3800 L la spin mE DNA concentration iii 2 filtrate 7 2 25 ug mL Step i Step2. 0 ARP DNA std B E 2 5 ARP DNA std E C m 5 ARP DNA std 10 ARP DNA std a om eee on N samples sample 11 sample 19 n sample 12 sample 20 i ARP DNA Standard Soln DNA Binding Solution 100 u r blank 60 u L TE Buffer 100 u L DNA Binding Solution DNA 60 i L 12 34567 89 10 11 12 1 250 u L well x5 2 HRP Streptavidin 150 u L well 3 250 u L well x5 4 Substrate Solution 100 u L well 2 0 10 20 30 40 Number of AP sites 100 000 bp 03 ARP DNA Standard Soln 0000000 0000 IvVnnan 1 0000 ed 597 0 2 DNAQ AP site OO DNA 1 LU LI LU I OO D ARP O DN 3 Filtration Tube DNA u u O oO D 00 D D D Oooo Ooo Ooo oO oO L3 L3 C3 r3 000000000 000000000 000000000 L3 O O O 000 Al oog O 0 O o HO Co L3 L3 L3 e p HBgH o goooogaoaon 25050 ppbaodo000000000 well 50 p LO 00000 1 mol 00000000 al O 0 O nmp O 0 O oOo 0000 0000 0000 pH O gt 3 HO Oooo Oooo o 2 000 00 00 D po0000000000 po00000000000 po00000000000 Oo oO Ono Ono uu 1 Sancar A and Sancar G B Annu Rev Biochem 57 29 1988 2 Lindahl T and Nyberg B Biochemistry 11 3610 1972 3 Liu
3. OOOO 29 E 11 DOOOOG00000015mLQ 0000000 OO ARPO O DNAD OO 00 S QUO0000 O OKtDO0do0o0o0oDo0 DNA D UU Urna O O TE Buffet 1 BH B i 0000000000000 DU TE Buffer 500 mL L1 O 500 mL Tris 606 mg 10 mmol L 3NA 206 mg 1 mmol L O O 6 mol L pg HCIO php 75600g8gpnagaggaganagnmnaa ug npagaggggugapnadgutttbtbnBda2asoox gti 5U00000000 O E O Filtration Tube 0 0000 00 DNAD OOO 90 00000000000 ARPO 0 DNAD O O 2 25 H g mLT O O O 2 AP site 1 O 1 81100000 ARPO O DNA 90 u LO 1 5 mi0 O 00000000 TE Buffer310 uO OOOO ARP DNA Standard Soln 1 well 9 9 60 u L 3 well OOOO00 20000 100000 ARPOO DNAD 1 well O 60 WL 3 well 1 L1 L1 LI NA Binding Solution 100 u LO DNAD OOO ell L C B 1 D 0000000000 00000 UU ut O O Washing Buffer DO OOOO0001L0 000000 PBSTOOO000 RP Streptavidin 9 O PBST 4 0000 0 DB 000 HRP Streptaviding 0 O O O HRP Streptavidin 10 u L PBST 40 mL well DODODODoO D UU UL UU U U PBST 250 n Ln DO d d C CL O 7 GU 7 CEU 0 welig uu LU U uu O well u ut O u uu 55 E AA O ODO E OL Ooo po000000 DU po000000 DU poo000005 D 8 9 10 O O HRP Streptavidin 150 u LO O well OO00 37 Q 1000000000000 6SOO000000000 PBSTOOOO0D0SOO D Substrate Solution 100 u 37 Q 1000000000000 pad gon 000000000 O00 6309 670 nmt 000000 OOOO LOO wel 0000 000000 000000 11 DNAD O AP sieg 00000000000 0 300 1 2 34 5 6 7 8 9 10 11 12 E
4. DOJINDO MOLECULAR TECHNOLOGIES INC Nucleostain DNA Damage Quantification Kit AP Site Counting For the Detection of the Number of Abasic Sites in Genomic DNA OO00 OOOOOY 20 samplen 0 DODODODODODODODNAODoDO DNA polymerase 0000 000000000000 D ARP DNA Standard Soin 000000000000000000000000 A ARP Solution PME uuu DNA Binding Solution 10mL x 1 uuu Washing Buffer 1 pack DNAQOOOOOODOOOOOOOOOOOOO DARAS 25 pL x 1 0000000000000 AP ste apurar Pee HE midnicsten 00 0000000000000000 I Filtration Tube 20 tubes O AP site 0 0 0 DNAG 1 Ba d Bb U G 7E CD 96 well Microplate U bottom 96 well x 1 u uuu Nucleostain DNA Damage Quantification Kit AP xm PONN d A a aA Site Counting 9 AP sie 000000 ARP U1 BE OU s Tm N Aminooxymethylcarbonyl hydrazino D biotin a Capo 0000000 D DNAL D B C 900000000000 ugmguggmgg mngguug COOOOOONAgOAPsiteQ 000000000 D o5ml t 5mL 000000 000000 Oooo lt a 000000000000 DNAD 0000 0 AP site o 0000000 ARP Standard DNA EH HL B D U U AOS copo 00000000000000 DNAD 0 0000000 DNAD AP sit D 000000 RNAD DI AP step 000000000000 DOBDpa Bog Do OD 0 0000000000000 RNase AJ 0 0 0 RNAD B B d DL OB 70 7 U 7 UI UI DNAJ ODO DD O Dg 0000 000000000 ugpnpnagguuautaab
5. i Step iii U 000 cup 1004 LJ TE buffer 000000 4004 Lg 2004 LO TE buffeqooodgddgggg 0200000 4004 LN ARD O O TE buffer 0 0 ARPO O OOOO ARO 04900000 ARPO O ENT O OM O Fil tration Tube 0 0 DNA 0 0 C D C D 2500xg OO15m900000000000000 2500xq 150 00 0 D 0100000 pnagaagnmnaua 90 uL ARPO DNAL soup 0000000 18 Cl 0000 LU D D D 310 pl TE buffer 00000 A standard soln well 000 0 ZO Vv 7 7 ARPO DNA 0 40 ARP DNA yopo300009000000000000 standard soln 100 uL DNA binding solution OOOO Hi weld 4 000000000000000 pod0000000 DNA Damage Quantification Kit DK02 General Protocol at a Glance Day 2 General Protocol L OO DO L UI U U UU U UU DO doad000S00000000wel 000000 pad0000000000000 D O HRP Streptavidin 150 uL O O well O O O 37 0 1000000000000 c 1 4000 7 0 1 HRP Streptaviding F1 L1 LU LU UL LU LU LLLULULULULUULULLILUL __10 uL HRP Streptavidin 40 mL 7 PBST IR EEE IES HH IEEE EHETHOHILEHTEHLELE EH EHEHITE Step 50000000000 PSNOOOOO0NOOU000 100 uL Substrate Solution wel OO 0 37 CO 1000000000000 0000000000000650m 000000000 DNAD D A site O po00d000000 90 0 6500000000000 00000000 0000wel 0 50uLQ 0000000000000 0 00500 1MOOO0000 40nmg 000000 000000000 DNA Damage Quantification Kit DK02
6. ndddtbNA rn O oooO DODBDODDODDOO DD ODO DNAQOD o oooO HOOO0000O DODOODODNADO ODO Dg 000000000 Get pure DNA Kit Agarose Get pure DNA Kit Blood Get pure DNA Kit Cell Tissue DNAJ O DDD Dog 0000 00000000 0120 489548 00000000000 Jl zen jarcinnun nnun f LI ie Pe EE TE Buffer 380p L L uuu t ket 00000 ARPO O DNA 400 p L 2 500x 9 151 O 00 2 25 u g mL Step 1 Step 2 Step 3 LH D cup 100u L TE Buffer LQ TE Buffer da zen o Y buf on ras 000 00000 af UU AQ 0000 iltration Tu x 1 5 mi O00 2500x 90 150000000 od er z ee REET CoC do000000Npo0000000 1 5mL 000000000 01 00 DNAD ARPO O n mit 1 0 0 DNAD ARPO DL 001000 1 O 000000 DNAD TE Buffet 0 0 0 OOOO 260 nmyr 00000000 0000 DNAD 0 O50 pgm oo00001 0000 10 po mLQOOOOUUUOTE Buffer 00000000 O O DNAD O 10 u LO ARP Solution 10 p LO 0 5 mUJOOOQUQU0U00000 37 C 1000 DOO 000000000 380 uL O TE Buffer 00000 4 QQ Filtration Tube cup 100 u LO TE Buffer 220000009000 U Filtration Tube 0 O00 5 00000 2 500x go158 0 00 00000007 O 6 5 Filtration Tube 400 u LO TE Buffer O OO 00000000000 ARPO O DbNAD D OOO uui V 300 00 7 DO000002500x 9015800000000 Dun 8 7 Filtration Tube 200 u LO TE Buffer O OO 00000000000 ARPOO DbNAD D OOO uui V 300 0 9 DODDODODNAJOD1s5mooo00000000 10 000 200 yu LO TE Buffer 8 Filtration Tube 00s900000000000000000ARP O O DNAD OO 80 DNAT 1
7. zzi M and Talpaert Borle M J Biol Chem 260 5252 1985 4 Weinfeld M Liuzzi M and Paterson M C Biochemistry 29 1737 1990 5 Chen B X Kubo K Ide H Erlanger B F Wallace S S and Kow Y W Mutat Res 273 253 1992 podo00000000 ATI D D B U 7I 7E DE E D UI 7 DI DE U D U 1 0 0 DNAQ OO000000 D DNA Binding Solution 000000000000 DNA Binding Solution Y 0 0O 0 DNADOOOUO OHOOUO00000 4 000 O 0 O O HRP Streptaviding 84000000000 ubstrate Solution 4000000 OOOO00d OOO P Streptavidin O0 0000000000000 uungaagngnaggaduttacld Streptavidin OO O A 00 5 Or d d b aat upaugnnanpnggnagagaguauutun ENMLILIL gr OI Ee sre ZU o EJ E Se ey wm Doon U DNAJO 0000 OOOO ONAQ OOO 100 p g mLE C ELE D DNAQDOOOUOOUUO DNAD OO ARP Solution 0000000 0 0 00 Filtration TubeQ OOOOUO000000 NAQ AP site 00 00 00 0 GO 1 100 000bpQ 1 QOOOO00d000g 97 90 IV ct jg CO gt D ba 5030000 ogi COO0 welQOO00000 1 HRP Streptavidin 0 0 TI 0 0 PBST 1 4 000 D000 00000000 HRP Streptavidin O0 00000000 20000000000 2 000 PBSTO O wei 500000000000 2pod0000 0000000000000 1 0 ARP DNA Standard Soln 400000000000 2000 PBSTO O welig Sr 0D00D00D000 30000000 PBSTD0000000000000PBST
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