ࡱ> EGD9 R3bjbj2@l$ "!!!!!!!$# /%2!!!B!!!! ˌmF!!!0"!a%a%!PCR8^;`Gl PCRNirv5ulhKme 00N,:N48hNQ gNg}YNS_e5ulhKm 'YN48hT&^W NĉRum1Y0 GP4'` NQsibXag&^ 00PCRS^vsQ.s g`$!jg8hxv6RY a$_irv(ϑNyr_'` b$vv(ϑS c$PCR_sagN0[~bSVN^[ NsۏLRgxvz0 00!jg`$!jg-N+T gBgˆ}v( a$!jg-N+T gTaqvb6RBR b$!jg-Nˆ}v(l gm SdQ yr+R/fgrSO-Nv~ˆ}v c$(WcS6RY!jge"N1YǏY b8TeQZ0d$!j g8hxS'` N{_^0(WvT_ir(ϑ}Ye NQsibX&^ g gS/fh,gvmSY t !jg8hxcSǏ zQNku V M6R gHe 3z[vmSYtm vQ z^N^ V[ N[af9e0 00v1Y;mfbcev bee$Nyv TeO(u NRg/f&TVvv;m'`'N1Yb NY [GP4'`0lav/f ge_RTaqvbnYN-0 00_ir_ir(ϑ0_irvSm^0$Nag_irvSm^/f&T[y /fPCR1Y%bibXag&^ N t`0[f%_cev8^SV0 gNybSv_irTb(ϑ g $Nag_irNagSm^ ؚ NagSm^NO bNOHesv N[yibX [V{:N`$ [N*N}Yv_irTbUS MO0a$_irvSm^ NN wOD

eQ{QυR [_irS(M1YHe0c$_ir NTt Y_ir^ NY _irKNb_bNZSOI{0 00Mg2+Sm^Mg2+yP[Sm^[PCRibXHesq_T_'Y Sm^ǏؚSMNOPCRibXvyr _'` Sm^ǏNORq_TPCRibXNϑuOPCRibX1Y% NQibXag&^0 00S^SOyv9eS8^ۏLPCRibXǑ(uvSOy:N20ul030ul050ul0b100ul ^(uY 'YSOyۏLPCRibX /f9hncyxT4N^hKm N Tvv [ (WZP\SOyY20ul T QZP'YSOye N[!j"}agN &TR[f1Y%0 00irtSVS'`[PCRibXegvS_͑ YS'`)n^NO S'`ew g gSQsGP4'`;kp)n^ǏNO S^yr_'`ibX MNOyr_'`ibXHeskp)n^Ǐؚq_T_irN!jgv~T MNOPCRibXHes0 ge؏ g_(uhQv)n^ hKmN NibXNb4lnQvS'`0kpT^8O)n^ ُ_N/fPCR1Y%vSVKNN0 00v^RS_Yv^RSuzSb:1Y q_T_irN!jgyr_'`~T bVv^Rg k:1YO_irN!jg1YSNe^R vQPCRibX/f NObRv0 GP3'` 00QsvPCRibXag&^Nvvv^Rag&^N gevQag&^fteP N^fؚ0 00_ir NT bvibX^RN^vvibX^R g Tn'` V (WۏLPCRibXe ibXQvPCRNir:N^vv'`v^R0v^R*Ywb_ir*Yw [fQsGP3'`0͑e_ir0 00v^RbibXNirvNSalgُyalg g$NySVN/fte*NWV~b'YGrkvNSalg [GP3'`0ُyGP3'`S(uN Nel㉳Qd\Oe^\_{g 2bk\v^R8TeQR7hgQbnQy_{Y0dvS Nؚ)nvir(Y @b gՋBRbhVPgGW^ؚSmk0@b(uy_{S7hۏg4YI{GW^N!k'`O(u0_e (WRh,gMR S^{TՋBR(u+}Y~gq\ N4xOWX[(Wv8hx0N/fzzl-Nv\Grk8hxalg ُN\Grkkv^Rw FO gN[v Tn'`0SNvbc N_irNeT SibXQPCRNir [GP3'`vNu S(u]_PCRelegQ{bmd0 Qs^yr_'`ibX&^ 00PCRibXTQsvag&^Nv'Y\ NN b'Yb\ b TeQsyr_'`ibX&^ N^yr_'`ibX&^0^yr_'`ag&^vQs vQSVN/f_irNv^R N[hQNe0 b_irZTb_bNZSO0N/fMg2+yP[Sm^Ǐؚ0kp)n^ǏNO SPCR_s!kpe ǏY gsQ0vQ!k/fvv(Tϑ __NNegnvvfQs^yr_ag&^ SNegnvv R NQs vϑǏY ge_NOQs^yr_'`ibX0vQ[V{ g_e͑e_ ir0QNOvϑbbcSNegnvv0MNO_irϑ S_XR!jgϑ Q\_s!k pe0S_cؚkp)n^bǑ(uN)n^pl(93!S'` 65!]SkpN^8O)0 QsGrrb&^bmb&^ 00PCRibX geQsmb&^bGrr&^b0Wk7h&^0vQSV__1uNvϑǏYbvv(ϑ ] dNTPSm^Ǐؚ Mg2+Sm^Ǐؚ kp)n^ǏNO _s!kpeǏY_w0vQ[V{ gQ\vϑ bbcSNegnvv0a$Q\dNTPvSm^0S_MNOMg2+Sm ^0XR!jgϑ Q\_s!kpe0 KQPCRNir 1)KQPCRNirvgOagN/fNHN gsOceQGrk}SOk[nx[011ceQGrk}SO 8^:NgsOk id\pek18b81_NL0^Km[k20-40݄e(uc[ek108!ke cfu/ ugaS`~ހ hf}SO1YST0S/fޏcvalgN8hxv0T4 DNAޏcvM1801 M1804 M1794 (ϑhQ}Ye8hxvalg N^(uvQ[egnvT4 DNAޏcvfbc0 D (upGEM-TbpGEM-T Easy}SO ޏcpGEM-Tck[gq lSؚsaS`~ހ108!ke cfu/ug , cgqc[v[ek S_100*Ñ= vQ-N60%^:N}ve YNu>20-40݄e, l g̃=b\ g̃= ޏc g0 4)[gq[~g}Y tSl gV6e0RvvGrk [QNNHN A ޏc(u[)nO)n1\e n'YYpeKQ :NcؚHes 4!ǏY0 B ceQGrk&^ galg O3`-T:1Y bb6Rޏc b6RlS0:Ndk \ceQGrkTpGEM-Tck[gqmT Qޏc0YMNON[gqṽ=pe ceQGrk~S b͑e6RY0YNu'Yϑv݄e ceQGrkalg g8hxv OpGEM-TbpGEM-T Easy}SO3`-T:1Y0 C ceQGrk NNޏc0(uQ~SvceQGrk VSUVǏ^gq\ e gSu0UVǏ^gq\ONu'VvUNZSO N)RNޏc DNA_͑e~S0 D &^ gO YRvpDNAZTvvibXNir+gzeA T/fpGEM-TbpGEM-T Easy}SOKQ@b0RTaq DNAZTvT8hxS(W+gzRA0`gpGEM-T pGEM-T Easy}SOb/gDeTM042 0 E ؚ^͑ Y^RSO N3z[ (WibX-NNu:1YT͑c YSsceQGrkؚs0WNu:1YT͑c (u͑~:w'YFg̃̃*h YSURE~ހ PCRS^SO|NS^agN hQvPCRS^SO| 000000 10ibXQm 0 10ul 000000 4ydNTPmTir 0 T200umol/L 000000 _ir 0000 0 T10^100pmol0 000000 !jgDNA 000 00.1^2ug0 0 00000Taq DNAZTv 0 2.5u0 000000 Mg2+000000 1.5mmol/L 000000 RSb N4l 0100ul 00PCRS^N } SRPCRS^vir(;N gNysS_ir0v0dNTP0!jgTMg2+ 00_ir _ir/fPCRyr_'`S^vsQ. PCR Nirvyr_'`SQN_irN!jgDNANev z^0t N SwSNUONk!jgDNA^R 1\ cvQNev[8hxZP_ir )R(uPCR1\S\!jgDNA(WSOY'YϑibX0 _ir^u_N NSR `$_ir^ 15-30bp 8^(u:N20bp]S0 a$_iribX荦^ N200-500bp:N[ yr[agN NSibX10kbvGrk0 b$_irxWG+C+TϑN40-60%:N[ G+C*Y\ibXHeg NsO G+CǏYfQs^yr_ag&^0ATGCg}Y:gR^ MQ5*NN Nv VdTb'VvU8hxvb2NcR0 c$MQ_irQQsN~~g MQ$Nag_irNe yr+R/f3'zvNe &TROb_b_irNZSO Nu^yr_vibXag&^0 d$_ir3'zvxW yr+R/fg+gSPpe,{N*NxW ^%Nf Tn'`0_irϑ kag_irvSm^0.1^1umolb10^100pmol NgNO_irϑNu@bv~g:N}Y _irSm^OPؚO_wMT^yr_'`ibX NSXR_irKNb_bNZSOv:gO0 00vSvQSm^0vMR g$NyTaq DNAZTvO^ Ny/fNhp4luFg̃-Nc~v)Y6qv SNy:N'ỸTbvWV] zv0PSNxQWvPCRS^~vϑ2.5U(c;`S^SOy:N100ule) Sm^ǏؚS_w^yr_'`ibX Sm^ǏNORTbNirϑQ\0 00dNTPv(ϑNSm^0dNTPv(ϑNSm^TPCRibXHes g[RsQ| dNTP|HT|r YOX[ NS_fS'`1YSuirf[;m'`0dNTPnmHTx'` O(ue^MbؚSm^T N1M NaOHb1M Tris0HCLvQm\vQPH0R7.0^7.5 \ϑRň -20!QQOX[0Y!kQOOdNTPM0(WPCRS^-N dNTP^:N50^200umol/L $\vQ/fla4ydNTPvSm^vI{( I{id\M6R) YvQ-NNUONySm^ N TNvQ[Qye(OPؚbOPNO) 1\O_wM0Sm^ǏNOSOMNOPCRNirvNϑ0dNTPNMg2+~T O8nyvMg2+Sm^MNO0 00!jg(vWV)8hx0!jg8hxvϑN~S z^ /fPCRb%N&TvsQ.sKNN O~vDNA~Sel8^Ǒ(uSDSTˆ}vvKegmSYth,g0SDSv;NR/f n~ހ Nv{|Nˆ}v( V n㉜ˆ}v 4xOW~ހ v^㉻y~ހ-Nv8hˆ}v SDS ؏Nˆ}v(~T lm ˆ}vvK4l㉈mSˆ}v( yr+R/fNDNA~Tv~ˆ}v Q(u g:gnBRZN/lNbccˆ}v(TvQ[~ހ~N (uYNb_Nlm8hx0cSv8hxsSS\O:N!jg(uNPCRS^0N,4N^hKmh,g SǑ(u_{Oveln~ހ ˆuSSO mSdSgrSOvˆ}v(OvWV8ny vc(uNPCRibX0RNA!jgcSN,Ǒ(u_kx0lx̀bˆ}vvKl 2bkRNaseMRNA0 00Mg2+Sm^0Mg2+[PCRibXvyr_'`TNϑ g>fWvq_T (WN,vPCRS^-N TydNTPSm^:N200umol/Le Mg2+Sm^:N1.5^2.0mmol/L:N[0Mg2+Sm^Ǐؚ S^yr_'`MNO Qs^yr_ibX Sm^ǏNOOMNOTaq DNAZTvv;m'` OS^NirQ\0 00PCRS^agNv b 00PCRS^agN:N)n^0eT_s!kpe0 00)n^Nevn WNPCRSt Nek nS'`-kp-^8O N*N)n^p0(WhQS^-NǑ(u N)n^pl SDNA(W90^95!S'` QŏQtS40 ^60! _irkpv^~T0Rv^R N 6qT_GS)n70^75! (WTaq DNA ZTvv\O(u N O_irl!jg^8O0[NwvWV(^:N100^300bpe)SǑ(uN)n^pl dS'`)n^Y0kpN^8O)n^STN:NN N,Ǒ(u94!S'` 65!]SkpN^8O(dk)n^Taq DNAvN gؚvPS;m'`)0 00`$S'`)n^NeS'`)n^NO N[hQ/f[PCR1Y%vg;NSV0N,`Q N 93!^94!minNO!jgDNAS'` NON93!R^e FO)n^ NǏؚ V:Nؚ)nsX[vv;m'` gq_T0dkek NOvWV!jgbPCRNir[hQS'` 1\O[PCR1Y%0 00a$kp( Y'`))n^Nekp)n^/fq_TPCRyr_'`v͑V }0S'`T)n^_QtS40!^60! SO_irT!jgSu~T01uN!jgDNA k_ir YBg_Y _irT!jgKNvxd~T:gO܏܏ؚN!jgNeKNvxd0kp)n^Ne SQN_irv^0xW~bSvQSm^ ؏ gvW^Rv^0[N20*N8hx G+C+Tϑ~50%v_ir 55!:N bgkp)n^vwp:Nt`0_irv Y'`)n^SǏN NlQ_.^R bTv)n^ 00000TmA\ay|78ILUWZ]`c  01>BQTXY~!"$(-]aimno~  01GHLS^`  1 2 6 < > C E I K P Y \ _ e h m w {            " & ( * . 1 4 6 8 = > U X Z ^ ` e h m s {       , / 5 8 @ F H J Z ^ z }        ! 8 : D F Y \ c f o r } ~     % & ) , 1 < A Q R V Y \ ] n p x y { |  !$'04BEY\_`tw| '(CE`adf AEPSW]^ $%(.0367:@DPT[_ceghqwxy}")*QUabefsv "#&:=CGJSUY\_`ilp-/0258=VWZ]^cdeno0369:<=JMacegy+/23679:>?AHIefhky{|  !-/02;=Rpsv}5<IMeh;>@DSXkpux|~ ).37EKSYbgor~lo$)16;>cgtx!059U[#'148;^csxKO'+W\pu{     W X u v } ~         F H | } ! + . O R e f = A n p  !$:@ijmq)/8=OT[_bdns!$)28:=`eJMFKEJQVhm{:<Y\jm}+/NRUZrvFL^c3333s33333333333333333333333333333s3333333333333333s3333333333333333333ss333333333s3sssssssss3sssss3ss33s33ss33333333s33333333333333333333333333333s33333s3333333333333333333333s333333333Rjm-C:\WIN98SE\Desktop\[el6eƖ\RP[uirf[\PCR8^;`Gl.docjm3C:\WIN98SE\Desktop\[el6eƖ\RP[uirf[\PCRb/g\PCR8^;`Gl.docjm3C:\WIN98SE\Desktop\[el6eƖ\RP[uirf[\PCRb/g\PCR8^;`Gl.docjm3C:\WIN98SE\Desktop\[el6eƖ\RP[uirf[\PCRb/g\PCR8^;`Gl.docjm3C:\WIN98SE\Desktop\[el6eƖ\RP[uirf[\PCRb/g\PCR8^;`Gl.docjm3C:\WIN98SE\Desktop\[el6eƖ\RP[uirf[\PCRb/g\PCR8^;`Gl.docjm@C:\WIN98SE\Application Data\Microsoft\Word\ ꁨRb` Y OX[PCR8^;`Gl.asdjm3C:\WIN98SE\Desktop\[el6eƖ\RP[uirf[\PCRb/g\PCR8^;`Gl.doc@zpUnknownG:Times New Roman5Symbol3& :Arial;[SOSimSun qhV˂fSfq )!-!),.:;?]}    & 6"0000 0 0 00000 =@\]^([{  0 0 00000;[22Qjmjm Oh+'0`   ( 4@HPXssjmmm Normal.dotjmm14mMicrosoft Word 9.0@q@LHK_@Ƈlq ՜.+,0 hp|  r)   Ŀ  "#$%&'()*+,-./012356789:;=>?@ABCFRoot Entry FˌmH1Table!a%WordDocument2@SummaryInformation(4DocumentSummaryInformation8<CompObjfObjectPoolˌmˌm  FMicrosoft Word ĵ MSWordDocWord.Document.89q