PCR Analyzer Troubleshooting: Cov lus nug nquag nug thiab daws teeb meem

Polymerase saw cov tshuaj tiv thaiv (PCR) cov tshuaj ntsuam xyuas yog cov cuab yeej tseem ceeb hauv molecular biology, tso cai rau cov kws tshawb fawb kom nthuav dav DNA rau cov ntawv thov xws li kev tshawb nrhiav caj ces mus rau kev kuaj mob. Txawm li cas los xij, zoo li txhua yam khoom siv nyuaj, PCR analyzer tuaj yeem ntsib teeb meem uas cuam tshuam rau nws txoj haujlwm. Kab lus no hais txog qee cov lus nug txogPCR tsom xamkev daws teeb meem thiab muab cov tswv yim daws teeb meem rau cov teeb meem tshwm sim.

1. Vim li cas kuv cov tshuaj PCR tsis ua kom nrov?

Ib qho ntawm cov teeb meem tshwm sim los ntawm cov neeg siv yog qhov tsis muaj peev xwm ntawm PCR cov tshuaj tiv thaiv kom nthuav dav DNA lub hom phiaj. Qhov no tuaj yeem raug ntaus nqi rau ntau yam:

Kev tsim primer tsis raug: Xyuas kom koj cov primers tshwj xeeb rau lub hom phiaj sib lawv liag thiab muaj qhov zoo tshaj plaws melting kub (Tm). Siv cov cuab yeej software rau primer tsim kom tsis txhob muaj kev sib khi tsis tshwj xeeb.

DNA Template tsis txaus: Xyuas kom tseeb tias koj siv tus qauv DNA txaus txaus. Tsawg dhau yuav ua rau tsis muaj zog lossis tsis muaj suab nrov.

Inhibitors hauv cov qauv: Cov kab mob hauv cov qauv tuaj yeem cuam tshuam cov tshuaj tiv thaiv PCR. Xav txog kev ntxuav koj cov DNA lossis siv lwm txoj kev rho tawm.

Kev daws: Txheeb xyuas koj tus qauv tsim qauv, ua kom cov qauv siab, thiab xyuas kom koj cov qauv tsis muaj cov tshuaj tiv thaiv.

2. Vim li cas kuv cov khoom PCR yog qhov tsis ncaj ncees lawm?

Yog tias koj qhov PCR cov khoom loj tsis yog raws li qhov xav tau, nws yuav qhia tau tias muaj teeb meem nrog cov tshuaj tiv thaiv lossis cov khoom xyaw siv.

Non-specific amplification: Qhov no tuaj yeem tshwm sim yog tias tus primer khi rau qhov chaw tsis xav tau. Txheeb xyuas qhov tshwj xeeb ntawm cov primers siv cov cuab yeej xws li BLAST.

Tsis yog Annealing Temperature: Yog hais tias qhov kub ntawm annealing tsawg dhau, tsis yog tshwj xeeb khi yuav tshwm sim. Optimization ntawm annealing kub los ntawm gradient PCR.

Kev daws: Paub meej qhov tshwj xeeb primer thiab ua kom zoo dua qhov kub thiab txias los txhim kho qhov tseeb ntawm cov khoom PCR.

3. Kuv tus PCR analyzer qhia cov lus yuam kev. kuv yuav ua li cas?

Cov lus yuam kev ntawm PCR analyzer tuaj yeem ceeb toom, tab sis lawv tuaj yeem muab cov lus qhia rau cov teeb meem tshwm sim.

Teeb meem Calibration: Xyuas kom tseeb tias PCR analyzer raug ntsuas kom raug. Kev saib xyuas tsis tu ncua thiab ntsuas ntsuas ntsuas yog qhov tseem ceeb kom tau txais cov txiaj ntsig raug.

Software Group: Qee zaum, software kab yuav ua rau muaj teeb meem. Pib dua koj lub computer thiab tshawb xyuas cov software hloov tshiab.

SOLUTION: Xa mus rau cov neeg siv phau ntawv qhia txog qhov kev ua yuam kev tshwj xeeb thiab ua raws cov kauj ruam daws teeb meem pom zoo. Kev saib xyuas tsis tu ncua tuaj yeem tiv thaiv ntau yam teeb meem.

4. Vim li cas kuv cov PCR cov tshuaj tiv thaiv tsis sib xws?

Cov txiaj ntsig PCR tsis sib haum tuaj yeem ua rau ntxhov siab rau ntau qhov laj thawj:

Reagent Quality: Xyuas kom meej tias tag nrho cov reagents, suav nrog enzymes, buffers, thiab dNTPs, yog tshiab thiab zoo. Cov reagents uas tas sij hawm los yog muaj kab mob tuaj yeem ua rau muaj kev hloov pauv.

Thermal Cycler Calibration: Kev ntsuas kub tsis sib haum tuaj yeem cuam tshuam rau txheej txheem PCR. Tsis tu ncua xyuas cov calibration ntawm thermal cycler.

Kev daws: Siv cov tshuaj zoo tshaj plaws thiab ntsuas koj lub thermal cycler tsis tu ncua kom ntseeg tau tias muaj txiaj ntsig zoo.

5. Yuav ua li cas txhawm rau txhim kho PCR cov tshuaj tiv thaiv?

Txhim kho qhov ua tau zoo ntawm PCR cov tshuaj tiv thaiv tuaj yeem ua rau muaj txiaj ntsig ntau dua thiab txhim khu kev qha.

Txhim kho cov kev mob tshwm sim: Kev sim siv ntau qhov sib txawv ntawm primers, qauv DNA thiab MgCl2. Txhua qhov tshuaj tiv thaiv PCR yuav xav tau cov xwm txheej tshwj xeeb rau kev ua haujlwm zoo.

Siv cov enzymes siab ncaj ncees: Yog tias qhov tseeb yog qhov tseem ceeb, xav txog kev siv lub siab ncaj ncees DNA polymerase kom txo tau qhov yuam kev thaum lub sijhawm ua kom nrov.

Kev daws: Ua qhov kev sim ua kom zoo kom pom cov xwm txheej zoo tshaj plaws rau koj qhov kev teeb tsa PCR tshwj xeeb.

Hauv cov ntsiab lus

Kev daws teeb meem aPCR tsom xamtuaj yeem ua haujlwm txaus ntshai, tab sis nkag siab txog cov teeb meem uas tshwm sim thiab lawv cov kev daws teeb meem tuaj yeem txhim kho koj qhov kev paub PCR. Los ntawm kev daws cov teeb meem no, cov kws tshawb fawb tuaj yeem txhim kho PCR cov txiaj ntsig thiab ua kom ntseeg tau cov txiaj ntsig zoo hauv kev siv tshuaj lom neeg molecular. Kev saib xyuas tsis tu ncua, ceev faj xaiv cov tshuaj reagents, thiab kev ua kom zoo ntawm cov tshuaj tiv thaiv kab mob yog cov yuam sij rau kev ua tiav PCR tsom xam.