??>? bd?a?9 R?@ibjbj2|?lXXXXXXXltttt,?l?n????????      $ ""P-!X?????-`XX??N```?ZX?X?`?`b`???hXX?? flt"3d0?C?r"8(r"`llXXXX?PCRS^SO|NS^agN -------------------------------------------------------------------------------- hQvPCRS^SO| 000000 10?ibXQm 0 10ul 000000 4ydNTPmTir 0 T200umol/L 000000 _ir 0000 0 T10^100pmol0 000000 !jgDNA 000 00.1^2ug0 0 00000Taq DNAZTv?0 2.5u0 000000 Mg2+000000 1.5mmol/L 000000 RSb N4l 0100ul 00PCRS^N } SRPCRS^vir(?N gNysS_ir0v?0dNTP0!jgTMg2+ 00_ir _ir/fPCRyr_'`S^vsQ.?PCR Nirvyr_'`SQN_irN!jgDNANev z^0t N SwSNUONk!jgDNA^R 1\ cvQNev[8hxZP_ir )R(uPCR1\S\!jgDNA(WSOY'YϑibX0 _ir^u_N NSR `$_ir^ 15-30bp 8^(u:N20bp]S0 a$_iribX荦^ N200-500bp:N[ yr[agN NSibX?0kbvGrk0 b$_irxWG+C+TϑN40-60%:N[ G+C*Y\ibXHeg NsO G+CǏYfQs^yr_ag&^0ATGCg}Y:gR^ MQ5*NN Nv VdTb'VvU 08hxvb2NcR0 c$MQ_irQQsN~~g MQ$Nag_irNe?yr+R/f3'zvNe?&TROb_b_irNZSO Nu^yr_vibXag&^0 d$_ir3'zvxW yr+R/fg+gSPpe,{N*NxW ^%Nf Tn'`0_irϑ kag_irvSm^0.1^1umolb10^100pmol NgNO_ir0ϑNu@bv~g:N}Y _irSm^OPؚO_w?MT^yr_'`ibX NSXR_irKNb_bNZSOv:gO0 00vSvQSm^0vMR g$NyTaq DNAZTvO^ Ny/fNhp4luFg̃-Nc~v)Y6qv?SNy:N'ỸTbvWV] zv?0PSNxQWvPCRS^~v?05U(c;`S^SOy:N100ule) Sm^ǏؚS_w^yr_'`ibX Sm^ǏNORTbNirϑQ\0 00dNTPv(?NSm^0dNTPv(?NSm^TPCRibXHes g[RsQ| dNTP|HT|r YOX[ NS_fS'`1YSuirf[;m'`0dNTPnmHTx?` O(ue^M?bؚSm^T N1M NaOHb1M Tris0HCLvQm\vQPH?R7.0^7.5 \ϑRň -20!QQOX[0Y!kQOOdNTPM?0(WPCRS^-N dNTP^:N50^200umol/L $\vQ/fla4ydNTPvSm^vI{( I{id\M?R) YvQ-NNUONySm^ N TNvQ[Qye(OPؚbOPNO) 1\O_w?M?0Sm^ǏNOSOMNOPCRNirvNϑ0dNTPNMg2+~T O8nyvMg2+Sm^MNO0 00!jg(vWV)8hx?0!jg8hxvϑN~S z^ /fPCRb%?N&TvsQ.sKNN O~vDNA~Sel?^Ǒ(uSDSTˆ}vvKegmSYth,g0SDSv;NR/f n~ހ Nv{|Nˆ}v(?V n㉜ˆ}v 4xOW~ހ v^㉻y~ހ-Nv8hˆ}v SDS ؏Nˆ}v(?~T lm ˆ}vvK4l㉈mSˆ}v(?yr+R/fNDNA~Tv~ˆ}v Q(u g:gnBRZ?N/lNbccˆ}v(TvQ[~ހ~N (uYNb_Nlm8hx?0cSv8hxsSS\O:N!jg(uNPCRS^0N,?N^hKmh,g SǑ(u_{Oveln~ހ ˆuSSO mSdSgrSOvˆ}v(?OvWV8ny vc(uNPCRibX0RNA!jgcSN,?u_kx0lx̀bˆ}vvKl 2bkRNaseMRNA0 00Mg2+Sm^0Mg2+[PCRibXvyr_'`TNϑ g>fWvq_T (WN,vPCRS^-N TydNTPSm^:N200umol/Le Mg2+Sm^:N1.5^2.0mmol/L:N[0Mg2+Sm^Ǐؚ S^yr_'`MNO Qs^yr_ibX Sm^ǏNOOMNOTaq DNAZTvv;m'` OS^NirQ\0 PCRS^agNv b 00PCRS^agN:N)n^0eT_s!kpe0 00)n^Nevn WNPCRSt Nek nS'`-kp-^8O N*N)n^p0(WhQS^-NǑ(u N)n^pl SDNA(W90^95!S'` QŏQtS40 ^60! _irkpv^~T0Rv^R N 6qT_GS)n70^75! 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(WTaq DNA ZTvv\O(u N O_irl!jg^8O0[NwvWV(^:N100^300bpe)SǑ(uN)n^pl dS'`)n^Y0kpN^8O)n^STN:NN N,?u94!S'` 65!]SkpN^8O(dk)n^Taq DNAvN gؚvPS;m'`)0 00`$S'`)n^NeS'`)n^NO N[hQ/f[PCR1Y%vg;NSV0N,`Q N 93!^94!lminNO!jgDNAS'` NON93!R^e FO)n^ NǏؚ V:Nؚ)nsX[vv;m'` gq_T0dkek NOvWV!jgbPCRNir[hQS'` 1\O[PCR1Y%?0 00a$kp( Y'`))n^Nekp)n^/fq_TPCRyr_'`v͑V }0S'`T)n^_QtS40!^60! SO_irT!jgSu~T01uN!jgDNA k_ir YBg_Y _irT!jgKNvxd~T:gO܏܏ؚN!jgNeKNvxd0kp)n^Ne SQN_irv^0xW~bSvQSm^ ؏ gvW^Rv^0[N20*N8hx?G+C+Tϑ~50%v_ir 55!:N bg?kp)n^vwp:Nt`0_irv Y'`)n^SǏN NlQ_.^R bTv)n^ 00000Tme\'`alg0fc^0 [h,gv~^BlNO? N?Ryukb~̃SW{Q~ހ DNA |6RTS;`RNAGWS\O:NibX!jg0Svc(u4N^h,gY@m0SOTm0mUm0kS0~ހ0;m~~I{|6RvDNAibXhKm0 ?PCRibXNirRg ??? PCRNir/f&T:Nyr_'`ibX vQ~g/f&TQnxS`?_{[vQۏL?NeQ{QυR [_irS(M?YHe0c$_ir NTt Y_ir^ N Y _irKNb_bNZSOI{0 00Mg2+Sm^Mg2+yP[Sm^[PCRibXHesq_T_'Y Sm^ǏؚSMNOPCRibXvyr_'` Sm^ǏNORq_TPCRibXNϑuOPCRibX1Y%? 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