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ProtocolofthepolymerasechainreactionfordetectinggeneticallymodifiedcomponentsinmaizeT~d$iB.BXj#r#r$iEfB#3;f~&HE03.1RSBBtransnene#@N*%&A%N,%NSi-F~fbillofllhc~sfi~DNA#9l~~~&jirp%AA&,~$!W@~~+~~~~,~@~SC$I~Wl~%~B~?$:E~3.2Bbl#&JJi$!polymerasechainreaction,PCR&&~~J?~'JYii%!~%l~?k&~~@~&DNA@fiH*M#S&HE&&{$T*@&&&BPJg2-t&1W+%~14@3)9II%8$5DNAH%#L&fiiW-BX$bIP9rJ2+BA~#X%6,f&#&DNAReB$~~8lTIJA~~~4~~#s4!(dNTP)%~&~~t&51~l~~~Mt$*~G~;f;II&~~~~%Ai#1~t~~-m6~@&rNtmsa~~L~nl~~~v49-3.3HMl%3.31PCRpolymerasechainreaction,j%j%PCR.33.2DNA,deoxyribonucleicacid,%&&#+&@.333dNTP,deoxyribonucIeos~detr~phosph~te.H#g@f('Z@@.33.4ATPPIdeoxyadeNOSineti.iphospl~~te,~~IB~@@3.3.5dCTP,deoxycyt~dlnetriphosph~te,a$f.fi#Z#@a33.6dtiTP,deoxyguaNOSinetriphoupliate,n#!&P583@mZll.33.7dTTP,deoxythym~dinetr1plrosphate,jttlffw8:ii@@~'33.8dUTP,deoxyurid~netriphosl)hate,MRtiirtfZ#@3.3.9bplbasepair,&4iH.3.3.10EDTAIethylenediaminetetraaceticacidtZ,Z@~Z,~.3.3.11TaglThermu~aquaticu,&!&%#!%.3.3.12Trisltris(hydroxymethyl)aminomethanetFi(~I&)$lBfe$2,3.3.13TEITris-C1,'EDTA##a,3.3.14IVR~invertaselgenefrommaize,3*&@fiBl%m.3.3.15Zeinlh*l#@biPBSB$,3.3.16CaMV35S135Spromoterfromcauliflowermosaicvirus,fi?g#%awJi$t#J355a3.3.3.17NPTIIlneomycin-3'-phosphotransferasegenet%#*-3'-@@8@&H.3.3.18NOS1terminatorofnopalinesynthasegene,861813U&9ElSm&&k+.3.3.19IVS21intronfromthemaizealcoholdehydrogenasegene,3#B)rB#%#%ElW&BJfiE2.3.320PATtphosphinothricinacetyltranrferasegene*PTf#Z,B%##Sfl.3.3.21HSP~O;O.8Kbintronfromthehsp70gene(heat-shockprotein),AQ%#j&@Q(hsp70)S$1BO0.8KbLBk4LX.3.3-22CryIA(b)1asyntheticgeneencodesthefirst648aminoa~ids~insecticidal-activeftruncatedproductidenticaltothatofCrylA(b)geneofBacillusthuringisnsi,subsp,%Z&~~fi~~&e~crylA(b)Sa.3.3.23CDPKIcalcium-dependentproteinkinase(CDPK)gene,$5%$&%&#@S$1.3.3.24Cry9CIinsecticidal-activeftruncatedproductidenticaltothatofCrgCgeneofBacillusthuringiensissubsp,~Z&i$~fi~&&~~l&lCrgCsm.3.3.25CaMV35SI135sterminatorofCaMVgenomcrtE~#i%Ri-~~SHl~35s%kF.3.3.26Pactin1Ithepromoterderivedfromtherice(Oryzasativa)actin1gene,A%Fh)/t~$j~EllLHI%&diStlf.3.3.27OTPIoptimizedtransitpeptidepene,fi#Si#&4i~.3.328mEPSPS,maize5-enolypyruvylshikimate-3-phosphatesynthase,$in*h*s(J#*mB&z-B%%!@.5.1nwEMSN/T1194rfiB&I%%%&E.5.2U##afl20gh*#fi,LF%%$II(lSOr?%PlfafiB2h)@120C.30min&Em3JfiSf&sN&&6$W$llZ#fig3SisS&flO.5mm%&A/]\.6.1QIX4#&&SBRDNA~~,~~X~S&H@QR@A~#NPBW~JL~R~~~~~I#~#~PCRfh*,4@3##VN5+~&@&DNA%@i*lHPCRrq@SW*#R#t;#db+t.'Bs%HSH&a.6.2OIRlMM~k4IP3RBWIBfi*XithBfPJ%J)~MiPt&#k)iZNtl)c%BMGB/T6682iBB'J-87k.26.2.2TaqDNAfiZ.$B6.2.3dNTPtdATP,dTTP,dCTP,dGTP,dUTP.6.2.4%111#I$%#.6.2.5a4kZB(EB)idt#f&3k&hrd.6.2.65#V%e'6.2.7%am.6.2.8SgJB.6.2.970%285.6.2.10S)FgMarker:50bp--300bp.6.2.11CTABSII)%6[13%CTAB(JB3if@)91,4401/L#4k4!10.2%(##N3)9k)189~Z,#.20mrnol/LEDTA,100mmol/LTrirHCllpH8.0.6.2.12TE@MaBit10mmol/LTris-HCllpH8.061mrnol/LEDTAlpH8.0.62.1310XP.CRaM?&1100rnmol/L,160mrnol/La@@.20mrnol/L,200rnmol/LTris-HCl(pH8.8)1%TritonX-10011rng/mLBSA.6.2.145XTBE@#*lTris54gl%@275g.0.5mol/LEDTA(pH8.0)20mLl$aa*31000mI,.6.2.15lOXk#@##eirO.25%@mlR,40%RM.6.2.16RNAR(10ug/mL).6.3B#6.3.1Mii##l@8lR@#.6.3.2~l~@Jift~b~.fiS/I\S~~GR1Mini+Aff,L&.6.3.3*#%%#IBWS9BCtWS#m#06.3.4Xvl&f0.001g.63.5REXH%ra.6.3.67L6WP##.6.3-7f%Qllk#ls,Xi%?k%g.6.3.8*i4.*%##.6.3.9fiJJbk#L.6.3.10b%HS$6#.6.3.11dg@P.6.3.12%@rWiX.6.3.13IBW.6.3.14PCR&MI$&.6.3.15@@%El3)#7l.X.6.3.16@if&l#,O.1pL-2pL.0.5pL-10pL,2PI,--20pL-10pL--100pL.20pL-200pL,200pL--1000pL.6.3.17#&$!1$#&%.6.3.18%N%XPCRW.6.3.19Eppendorf@1.5rnI,--5mL.6.3.20PCRI200pI-,500pI.n#B#.6.4aWJ@I6.4.1#faDNA%%2g$#T10mLddG$Fq,&A5mLCTAB?8YM%(?%BBtRNAB),?fi33,60Cdcb@B4#B1hr2000r/minF1$lL-5minrl8[k.#%t):,b~%#@S#VB/%dtBj[(#H3)91(1r241l)tEft,@f5min.8000r/minBIG5~~~~/I\JL@RI~~.SB%~~~~R~#RZNVG/#I#BS~(#B~)@~~~11)#3Ql#R5min18000r/rninRbb5minr@&~bk#?&b~0.65f$f$$~#J%~i@,133,12000r/rnin4CI$,L10minrfPk#%,&500pL70%Irf(LBP&$#-&,12000r/min4C'FIBbb5rninrBk.#~,#$E@I$rlf.,fmA50PI.TE,##FBB(4Caf&,d37'C'f%E!1h)I&@%,&DNA@@m.&~H.)fl&Stifi#DNA&fetH&J&@JBtDNA,6.4.2PCRW#64.2.1PCRE&#IR#rlhlwm%rn+fi,m-ix~mPCP.iz~~~u~2.~+~mm~~s~ti+~fi~g~uss~~rnljte4;n~rnpa*aafi~rnt~~t~~,~t~sm~#~~~~xt~,ul)c{t~~.e~tiiDNA#%9$XQMWI.2@SB39ki4.9blaPi4&1MPCREP#S6.4.22PCREh!lfRZ$#@94Cfi493'&2mln.94C$#40s,55f-58Cajk60s(R##Agj%j+Jil$!1,72C@#60S,35+3WC4;.72Cde4$5mi.4Ci51@.thSfl&!#;I~HM2&BVWt!ZX4PCREih?t8564111[#%53Rd&.64.3PCWrI'#P#Jr$%&w8]&%!3!I+%(lXTBE&TAE)$+J$2%HR8$Rllbf@($&?%55'C-6oC&libflAEHd!Jti&!#$tPJH@#t8€%0.5pg/mI.,-l&~?%~~~~~~~f3.14101'1.-15PI.PCRV#P%3)%l]1#12PI.k.#@@383a&,j&fifi#.81100bpI.addcrI)NAMarker&&GesHDNAMarker#j).?S&jiZ.3V/cm--5V/cmtflEE,&$J20min-40mln.~Ji!2~~~211R~#3)EiZ~D65aR#gi65.1AIIEiN4bI8]et.~S3i#fiNMIVR$M(ljtZein&f4)i(;li~h9~l%%~lXXDNABRkRkitt37PCRNN,It!k~Bflfl8~fkMJ!fl@FfB!#~%&?&TY$Ib226bp($173t~p)b!JPCKPCIstiRITiAPCRP#!rNI@J#,f!JlDNAB&BS.fiFlH,&DNA@4j!?<lG~#dPC'REGUJ~~?~~,$P~&@IDNA,BPJB@ltlBPcRPN.6.5.29bNaBlk!lWxfDNA@Ifd%BififiBJ&BWJI'CKMfit:,hll#IIAftXtRfl#l51Q%$Rfl*lIlX%rM*%t):,5iitN%*IOXYCRakfl(M13a)dNTP($dUTF)'l'aqUNG81.31%i#lDNAEm$IIE!A!#%$&i&lPJacJf~#@X#~m%.f;Wff~&&.&#%%fi~%isePeraCJltsdrtlt'-25mmol/I.2.5mniol/l.-5U/