Amava olawulo lweeprojekthi ezininzi kwaye enye ukuya kwimodeli yomboneleli othile yenza ukubaluleka okukhulu konxibelelwano lombutho kunye nokuqonda kwethu lula oko ukulindeleyo kwiFactory eyenziwe ngentengiso eshushu yaseTshayina ngokuSebenzisa okuPhezulu kunye noZinzo lwe-Ivd lokuSebenzisa iNyathelo elinye Probe Rt-QpcrKit V2, Besijonge phambili ekwakheni amakhonkco alungileyo kwaye aluncedo nabo bonke ababoneleli kwiplanethi.Siyakwamkela ngokufudumeleyo ukuba uqhagamshelane nathi ukuze uqale iingxoxo malunga nokuba sikwazi njani ukwenza oku.
Amava olawulo lweeprojekthi ezininzi kwaye enye ukuya kwimodeli ethile yomboneleli yenza ukubaluleka konxibelelwano lombutho kunye nokuqonda kwethu ngokulula okulindelekileyoChina Taq DNA Polymerase, Qpcr, Bayimodeli eyomeleleyo kwaye bakhuthaza ngokusebenzayo kwihlabathi liphela.Ungaze unyamalale imisebenzi emikhulu ngexesha elikhawulezayo, kunyanzelekile ukuba ulungele iimfuno zakho zomgangatho olungileyo.Ukukhokelwa ngumgaqo woBuchule, ukuSebenza, iManyano kunye nokuTshintsha.iqumrhu.ake i imizamo ebalaseleyo ukwandisa urhwebo yayo ngamazwe, ukuphakamisa umbutho wayo.rofit kwaye inyuse isikali sayo sokuthumela ngaphandle.Siqinisekile ukuba sele siza kuba nethemba eliqaqambileyo neliza kusasazwa kulo lonke ihlabathi kwiminyaka ezayo.

Iinkcukacha

50×20μl rxns, 200×20μl rxns, 1000×20μl rxns, 2000×20μl rxns

I-2X Real PCR Easy TM Mix-Taqman ebonelelwa yi-Real Time PCR Easy TM-Taqman kit yinkqubo entsha ye-premix esebenzisa i-probes ethile ye-fluorescent ye-Real Time PCR yokusabela kwe-amplification, enokuphucula kakhulu imveliso ethile kunye novelwano lokuphendula.I-ROX inikezelwa njengedayi yolawulo lwangaphakathi.

I-2X Real PCR Easy TM Mix-Taqman iqulethe i-Foregene ekhethekileyo eshushu-isiqalo seTaq DNA Polymerase.Xa kuthelekiswa nee-enzymes ze-Taq eziqhelekileyo, ineengenelo zokusebenza kwe-amplification ephezulu, isakhono esithile esinamandla sokukhulisa kunye nesantya esisezantsi sokungahambelani.Inokunciphisa ukukhuliswa okungangqaliyo kunye nokuphucula ukuchaneka kwe-PCR.

Amacandelo ekhithi

Iimpawu&izinto eziluncedo

■ Ilula—I-2X PCR Mix ukunciphisa impazamo yovavanyo kunye nexesha lokusebenza

■ I-Specific-optimized buffer kunye ne-hot-start Taq enzyme inokuthintela ukukhulisa okungangqalanga kunye nokwakheka kwe-primer dimer.

■ Uvakalelo oluphezulu-lukwazi ukubona iikopi ezisezantsi zetemplate

■ Ukuguquguquka okuhle—okuhambelana nezixhobo ezininzi zePCR zexesha lokwenyani

Isicelo seKit

Uhlalutyo lwe-qPCR

Ukuhamba komsebenzi

Umzobo

Ugcino kunye nobomi beShelf

Le khithi kufuneka igcinwe kude nokukhanya kwaye kufuneka igcinwe ku -20 ℃.Ukuba isetyenziswa rhoqo, inokugcinwa kwi-4 ℃ ixesha elifutshane (iintsuku ezili-10) .Amava olawulo lweeprojekthi ezininzi ngokwenene kunye nemodeli enye yomboneleli othile yenza ukubaluleka okukhulu konxibelelwano lwentlangano kunye nokuqonda kwethu lula ukulindela kwakho kwiFactory eyenziwe eshushu-intengiso yaseChina eFanelekileyo ePhakamileyo kunye noZinzo kwi-Ivd Sebenzisa iNyathelo elinye Probe Rt-QpcrKit V2, Besijonge phambili ekwakheni amakhonkco alungileyo kwaye aluncedo nabo bonke ababoneleli kwiplanethi.Siyakwamkela ngokufudumeleyo ukuba uqhagamshelane nathi ukuze uqale iingxoxo malunga nokuba sikwazi njani ukwenza oku.
Umzi-mveliso wenze intengiso eshushuChina Taq DNA Polymerase, Qpcr, Bayimodeli eyomeleleyo kwaye bakhuthaza ngokusebenzayo kwihlabathi liphela.Ungaze unyamalale imisebenzi emikhulu ngexesha elikhawulezayo, kunyanzelekile ukuba ulungele iimfuno zakho zomgangatho olungileyo.Ukukhokelwa ngumgaqo woBuchule, ukuSebenza, iManyano kunye nokuTshintsha.iqumrhu.ake i imizamo ebalaseleyo ukwandisa urhwebo yayo ngamazwe, ukuphakamisa umbutho wayo.rofit kwaye inyuse isikali sayo sokuthumela ngaphandle.Siqinisekile ukuba sele siza kuba nethemba eliqaqambileyo neliza kusasazwa kulo lonke ihlabathi kwiminyaka ezayo.

Akukho miqondiso yokukhulisa

I-1.I-Taq DNA Polymerase kwikiti ilahlekelwa ngumsebenzi wayo ngenxa yokugcinwa okungafanelekanga okanye ukuphelelwa yisikhathi kwekiti.
Ingcebiso: Qinisekisa imiqathango yokugcina ikhithi;yongeza kwakhona imali efanelekileyo ye-Taq DNA Polymerase kwinkqubo ye-PCR okanye uthenge i-Real Time PCR Kit kwimifuniselo ehambelanayo.

2.Zininzi ii-inhibitors ze-Taq DNA Polymerase kwi-template ye-DNA.
Ingcebiso: Hlaziya itemplate okanye unciphise inani letemplate esetyenzisiweyo.

3.I-concentration ye-Mg2 + ayifanelekanga.
Isincomo: I-Mg2 + yoxinaniso lwe-2× Real PCR Mix esibonelela ngayo yi-3.5mM.Nangona kunjalo, kwezinye iiprimers ezikhethekileyo kunye neetemplates, ugxininiso lweMg2 + lunokuba phezulu.Ke ngoko, unokongeza ngokuthe ngqo i-MgCl2 ukongeza i-Mg2+ yoxinaniso.Kuyacetyiswa ukuba kwandiswe i-Mg2+ 0.5mM ixesha ngalinye ukwenzela ukuba kuphuculwe.

4.Iimeko zokukhulisa i-PCR azifanelekanga, kwaye ukulandelelana kwe-primer okanye ukugxininiswa akufanelekile.
Isiphakamiso: qinisekisa ukuchaneka kokulandelelana kwe-primer kwaye i-primer ayizange ithotywe;ukuba isignali yokukhulisa i-amplification ayilungile, zama ukuthoba iqondo lokushisa le-annealing kwaye ulungelelanise i-primer concentration ngokufanelekileyo.

5.Isixa setemplate sincinci okanye sininzi kakhulu.
Isincomo: Yenza ithempleyithi yohlengahlengiso lodidi, kwaye ukhethe itemplate yoxinaniso ngeyona mpembelelo ilungileyo yePCR kulingo lwexesha lokwenyani lwePCR.

I-NTC inexabiso eliphezulu kakhulu le-fluorescence

Ungcoliseko lwe-1.Reagent olubangelwa ngexesha lokusebenza.
Isincomo: Faka indawo ngezinto ezintsha zokuvavanya ixesha lokwenyani lePCR.

2.Ungcoliseko lwenzekile ngexesha lokulungiselela inkqubo yokusabela kwe-PCR.
Isincomo: Thatha imilinganiselo yokukhusela eyimfuneko ngexesha lokusebenza, njengale: ukugqoka iiglavu ze-latex, usebenzisa i-pipetti tip kunye nesihlungi, njl.

3.Ii-primers zihlanjululwe, kwaye ukuthotywa kwee-primers kuya kubangela ukukhulisa okungangqalanga.
Ingcebiso: Sebenzisa i-SDS-PAGE i-electrophoresis ukubona ukuba iiprimers zithotyiwe, kwaye ubeke endaweni yazo ngeeprimer ezintsha zeLixesha loNyaniso lwemifuniselo yePCR.

Primer dimer okanye non-specific amplification

I-1.I-concentration ye-Mg2 + ayifanelekanga.
Isincomo: I-Mg2 + yoxinaniso lwe-2 × Real PCR Easy TM Mix esibonelela ngayo yi-3.5 mM.Nangona kunjalo, kwezinye iiprimers ezikhethekileyo kunye neetemplates, ugxininiso lweMg2 + lunokuba phezulu.Ke ngoko, unokongeza ngokuthe ngqo i-MgCl2 ukongeza i-Mg2+ yoxinaniso.Kuyacetyiswa ukuba kwandiswe i-Mg2+ 0.5mM ixesha ngalinye ukwenzela ukuba kuphuculwe.

2.Iqondo lobushushu le-PCR liphantsi kakhulu.
Ingcebiso: Yongeza iqondo lobushushu le-PCR nge-1℃ okanye nge-2℃ ngexesha ngalinye.

3.Imveliso yePCR inde kakhulu.
Isincomo: Ubude be-Real Time PCR imveliso kufuneka ibe phakathi kwe-100-150bp, ingabi ngaphezu kwe-500bp.

4.Ii-primers zihlanjululwe, kwaye ukuchithwa kwee-primers kuya kukhokelela ekubonakaleni kwe-amplification ethile.
Ingcebiso: Sebenzisa i-SDS-PAGE i-electrophoresis ukubona ukuba iiprimers zithotyiwe, kwaye ubeke endaweni yazo ngeeprimer ezintsha zeLixesha loNyaniso lwemifuniselo yePCR.

I-5.Inkqubo ye-PCR ayifanelekanga, okanye inkqubo incinci kakhulu.
Ingcebiso: Inkqubo yokusabela ye-PCR incinci kakhulu iya kubangela ukuchaneka kobhaqo ukuba kwehle.Kungcono ukusebenzisa inkqubo yokusabela ekhuthazwa sisixhobo sobungakanani bePCR ukuphinda usebenzise umfuniselo weXesha lokwenyani wePCR.

Ukuphindaphinda okulambathayo kwamaxabiso obungakanani

1.Isixhobo asisebenzi kakuhle.
Isiphakamiso: Kunokubakho iimpazamo phakathi komngxuma ngamnye we-PCR wesixhobo, okubangela ukuveliswa kakubi ngexesha lokulawula ubushushu okanye ukufumanisa.Nceda ujonge ngokwemiyalelo yesixhobo esihambelanayo.

2.Ubunyulu besampula ayilungile.
Isincomo: Iisampulu ezingcolileyo ziya kukhokelela ekuveliseni okungahambi kakuhle kovavanyo, okubandakanya ukucoceka kwetemplate kunye neeprimers.Kungcono ukuhlambulula itemplate, kwaye i-primers ihlanjululwe kakuhle yi-SDS-PAGE.

I-3.I-PCR yokulungiselela inkqubo kunye nexesha lokugcinwa lide kakhulu.
Ingcebiso: Sebenzisa inkqubo ye-PCR yeXesha lokwenyani yovavanyo lwe-PCR ngoko nangoko emva kolungiselelo, kwaye ungayishiyi ecaleni ixesha elide.

4.Iimeko zokukhulisa i-PCR azifanelekanga, kwaye ukulandelelana kwe-primer okanye ukugxininiswa akufanelekile.
Isiphakamiso: qinisekisa ukuchaneka kokulandelelana kwe-primer kwaye i-primer ayizange ithotywe;ukuba isignali yokukhulisa i-amplification ayilungile, zama ukuthoba iqondo lokushisa le-annealing kwaye ulungelelanise i-primer concentration ngokufanelekileyo.

I-5.Inkqubo ye-PCR ayifanelekanga, okanye inkqubo incinci kakhulu.
Ingcebiso: Inkqubo yokusabela ye-PCR incinci kakhulu iya kubangela ukuchaneka kobhaqo ukuba kwehle.Kungcono ukusebenzisa inkqubo yokusabela ekhuthazwa sisixhobo sobungakanani bePCR ukuphinda usebenzise umfuniselo weXesha lokwenyani wePCR.