Hauv PCR (polymerase cov tshuaj tiv thaiv) kev sim, ntim khoom yuav zoo li yooj yim, tab sis siv tau ntawm kev siv, thiab txhim khu kev qha cov txiaj ntsig tau. Txawm hais tias khaws cia thiab thauj lab {{1} npaj reagents lossis ua lag luam premixes tau tsim nyog thiab txhim kho kev sim ua haujlwm. Hauv qab no, peb qhia cov tswv yim tswv yim ntawm kev xaiv cov ntaub ntawv, ntim cov tswv yim, thiab cov ncauj lus cia.
I. Ntim Khoom Siv: Ua ntej ua ntej sib raug zoo thiab kev tiv thaiv
PCR cov kev coj ua yog rhiab rau qhov kub thiab txias, teeb, thiab cov khoom ntim khoom ntim yuav tsum tau raws peb qhov tseem ceeb: lub teeb {{{} Cov ntaub ntawv pov thawj.
Rau centrifuge tub thiab pcyprasylene (pp) yog qhov zoo dua qub rau kev hloov pauv nrawm dua, thaum 0.5 ML Rau 96 {- PC Daim Phiaj, peb pom zoo xaiv ib qho kev tsim qauv (rau qhov yooj yim ntawm kev tsim nyog ntawm kev tsim qauv (rau kev txuag chaw).
Txoj Kev Sealing: Cov leeg centresal centrifuge siv tiaj tus lossis tsa cov kaus mom (tiv thaiv kom tiv thaiv kom qhuav). Txawm li cas los xij, rau ntev - sij hawm cia, cua sov}- Sealing zaj duab xis (xws li sib npaug ntawm sab hauv thiab sab nraud siab. PCC cov phiaj yog pom zoo kom siv adhesive aluminium ntawv ci sealers (siab} {kua nplaum - siv).
Kev tiv thaiv lub teeb: reagents muaj lub teeb -} Cov khoom siv rhiab (muaj los ntawm qee cov tuam txhab) yuav tsum tau siv.
Ii. Kev kawm aliquoting: kev poob qis ntawm kev rov ua khov khov -} thahaw cycles
Aliquoting Reagents yog cov kauj ruam tseem ceeb hauv pCR ntim thiab ncaj qha cuam tshuam kev sim ua kom tiav.
Master Mix aliquoting: xws li Taq enzyme thiab DNTE} 10% npoo (piv txwv li, rau 20 μL tiv thaiv, AliTot 22 μl ib lub raj). Zam txhob siv ncaj qha los ntawm kev ntim khoom ncaj qha, zoo li rov ua kom khov (Taq enzyme yuav ua rau muaj kwv yees li 5% kev ua ib qho kom khov-thaw.
Primers thiab cov qauv: Primers (tshwj xeeb tshaj yog cov ntawv tso cai kev cai) tau pom zoo los ntawm 100 μm Tshuag daws teeb meem thiab khaws cia ntawm {}} 20 degree. Dilute rau kev ua haujlwm ua haujlwm (feem ntau 0.1-1 μm) ua ntej siv. Cov qauv xws li DNAtic DNA yuav tsum tau aliquoted hauv ib qho - siv cov noob kom tsis txhob muaj ntau.
Kev tiv thaiv cov lus qhia tsis zoo: Ua kom cov lus qhia txias ua ntej los tiv thaiv kab mob khaub ncaws ntxhua khaub ncaws, thiab hnav hnab looj tes thoob plaws tag nrho cov txheej txheem kom tsis txhob muaj cov tawv nqaij DNA.
III. Labeling thiab Recordkeeping: zam lub pob "dub" cuab
Caws daim ntawv cim yog qhov tseem ceeb rau cov pcR ntim khoom; Cov ntawv sau tsis yog tuaj yeem rhuav tshem tag nrho cov kev sim.
Yuav tsum tau cov ntaub ntawv sau npe: reagent lub npe (piv txwv li, "mm,"), "10 xyoo
Daim ntawv sau npe tshwj xeeb: Yog tias lub reagent muaj glycerol (antesrrower), dyes (piv txwv, "muaj 50%," muaj 50%
Sau cov synchronization: nws raug nquahu kom siv cov ntaub ntawv los yog xws li cov raj ntsuas (piv txwv li tus nqi tm) thiab koom nrog tus lej cim lub cev.
IV. Cia thiab Tsheb Thauj Mus Los: Kev Ntsuas Kub thiab Kev Tiv Thaiv Lub Cev
Qhov kev ruaj ntseg ntawm PC reagents yog heev nyob ntawm kev cia khoom, thiab kev tiv thaiv ntxiv yog xav thaum caij tsheb thauj mus los.
Kev Cia:
Enzymes (TAQ, Rov Qab Transcriptase): ntev {}}}}}}}}}}
Primers / probes: Khw ntawm -20℃rau ntau tshaj 1 xyoo (yog tias muaj cov pab pawg hloov kho xws li biotin, tiv thaiv los ntawm lub teeb);
Tsis - Enzyme Buffers (piv txwv li, PBS): Luv {}} sijhawm cia ntawm 4℃(txog 1 hlis).
Tsheb thauj mus los:
Txog kev hloov chaw txias, siv dej khov txias (- 78 degree) rau cov enzymes (ib ncig 0 degree) rau buffers rau kev thauj mus los luv luv.
Zam kev co kev co ntau dhau (PCR daim phiaj yuav tsum tau ruaj ntseg hauv cov thawv ua npuas ncauj kom tiv thaiv kev sib xyaw ntawm cov tshuaj reagents ntawm cov dej hiav txwv).
Thaum lub caij kub siab txias, ntxiv ib txheej insulating rau lub ntim (piv txwv li txhuas cov ntaub ntawv rasulation lub hnab + lub kas fes pob).
V. Cov teeb meem thiab kev daws teeb meem
Qhov Teeb Meem 1: Reagent nag lossis DNTPS lossis MGCL₂ Zej Zog ntawm Tsawg Kub (1000 RPM, 1 feeb) ua ntej kev tsis ncaj ncees. Dissolve nyob rau hauv 37℃incubator ua ntej siv thiab vortex kom sib xyaw kom huv si.
Teeb meem 2: Phaj Fealer Yooj yim dua Poels tawm → Resistant Sealer nrog tus nplaum-}} kev sim ua ntej).
Qhov Teeb Meem 3: Kev Siv Cov Ntawv Cog Lus Blog → Kev siv lub cim dej tsis huv (xws li lub ntsej muag dej (xws li lub ntsej muag tsis muaj dej) lossis laser -}}}}}} luam tawm cov ntawv sau los ntawm kev siv dej cawv.
Tag
PCR ntim yog ntau tshaj li "ntim" reagents; Nws yog thawj kab ntawm kev tiv thaiv rau kev sim siab kev cia siab. Los ntawm kev xaiv cov ntaub ntawv tsim nyog, ua rau cov txheej txheem aliquing, thiab tswj cov kev tswj kom sov thiab siv zog ua kom ruaj khov thiab muaj cov txiaj ntsig zoo. Nco ntsoov: cov ntsiab lus txiav txim siab ua tiav. Cov qauv pr kev tsim nyog tuaj yeem txuag lub sijhawm muaj kev daws teeb meem thoob plaws hauv txhua qhov kev sim!