PCR thev naus laus zis zoo ib yam li cov txheej txheem rov ua dua tshiab ntawm DNA, thiab nws qhov tshwj xeeb tso siab rau oligonucleotide primers ntxiv rau ob qhov kawg ntawm lub hom phiaj ib ntus. PCR muaj peb theem pib cov tshuaj tiv thaiv: denaturation, annealing, thiab extension. Cov txheej txheem tshuaj tiv thaiv no yog ua nyob rau hauv lub thawv tshuaj tiv thaiv PCR. Nrog rau kev txhim kho txuas ntxiv ntawm cov cuab yeej siv noob caj noob ces, lub thawv tshuaj tiv thaiv kuj tau ntsib qhov kev loj hlob zuj zus ntawm cov raj centrifuge los ntawm 1.5ml mus rau 0.2ml (0.25ml) mus rau nyias-walled {{ 7}}.2ml mob siab rau PCR. , 0.1mlpcr tub. Nrog rau qhov nce ntawm PCR amplification tshwm sim, high-throughput gene amplification ntim rau PCR strips thiab PCR daim hlau yog maj mam tsim.
PCR muaj peb theem pib cov tshuaj tiv thaiv: denaturation-annealing-extension
Denaturation ntawm DNA template:
Tom qab lub template DNA yog rhuab mus txog 94 degree rau ib lub sij hawm ntawm lub sij hawm, ob-stranded DNA ntawm template DNA los yog ob-stranded DNA tsim los ntawm PCR amplification yog dissociated, yog li ntawd nws yuav tsum tau ua ke nrog primer los npaj. rau qhov kev tawm tsam tom ntej.
Annealing (renaturation) ntawm template DNA nrog primers:
Tom qab cov qauv DNA yog denatured rau hauv ib qho strand los ntawm cua sov, thiab qhov kub thiab txias yog qis dua li ntawm 55 degree, cov primers tau ua ke nrog cov sib txuas ntxiv ntawm tib txoj hlua ntawm cov qauv DNA.
Extension ntawm primers:
Raws li qhov kev txiav txim ntawm Taq, DNA template-primer conjugate siv dNTP raws li cov khoom siv raw cov ntaub ntawv los ua ke ib qho tshiab semi-reserved replication saw ntxiv rau cov qauv DNA saw raws li lub hauv paus ntawm kev sib koom ua ke thiab ib nrab replication.
Basic Principles of PCR Technology
Cov thev naus laus zis tso siab rau cov tshuaj tiv thaiv enzymatic ntawm DNA polymerase nyob rau hauv muaj cov qauv DNA, primers thiab plaub deoxyribonucleotides.
DNA polymerase siv ib leeg-stranded DNA raws li ib tug qauv los pib synthesis nrog ib tug me me ntawm ob-stranded DNA. Ib los yog ob qho hluavtaws oligonucleotide primers tau ua ke nrog kev sib txuas ntxiv hauv cov qauv DNA ib leeg los ua ib feem ntawm ob-stranded DNA.
Nyob rau hauv ib qho chaw sov thiab ib puag ncig zoo, DNA polymerase ntxiv deoxymononucleotide rau 3'-OH kawg ntawm cov primer, thiab siv qhov no los ua qhov pib taw tes txuas ntxiv mus raws li 5'→ 3' kev taw qhia ntawm tus qauv los tsim cov DNA tshiab ntxiv strand.
Qhov sib txawv ntawm PCR raj thiab centrifuge raj
PCR tubes:
PCR cov tshuaj tiv thaiv phaj yog 96-zoo lossis 384-zoo, uas yog tsim tshwj xeeb rau cov tshuaj tiv thaiv batch. Lub hauv paus ntsiab lus yog tias qhov kev xa tawm ntawm lub tshuab PCR thiab cov sequencer feem ntau yog 96 lossis 384.
Centrifuge tube:
Lub raj centrifuge tsis tas yuav yog PCR raj. Muaj ntau ntau hom centrifuge raj raws li lawv lub peev xwm. Cov khoom siv feem ntau yog 1.5ml, 2ml, 5ml, 15 lossis 50ml, thiab qhov tsawg tshaj plaws (250ul) tuaj yeem siv los ua PCR raj.
Yuav xaiv PCR raj li cas
Peb tau xaiv tso cov PCR raj rau hauv kev cia siab ntawm kev xaiv qhov tseeb tshaj plaws rau qhov kub thiab txias.
Qhov tseeb tshaj plaws ntawm qhov ntsuas kub yuav tsum yog txoj haujlwm saum toj no qhov ntsuas kub hauv qab ntawm lub module (tsis hais qhov ntsuas kub tsis raug ntawm qhov ntsuas).
Txoj hauj lwm ntawm qhov ntsuas kub ntawm cov cuab yeej PCR sib txawv.
Piv txwv li, AB's 2720 tsuas muaj ib qho hauv nruab nrab, MJ's 200 muaj peb lub sensors, uas yog sab laug, nruab nrab thiab sab xis, Eppendorf's yuav tsum nyob rau hauv kab A lossis H, thiab Dongshenglong 811 muaj peb qhov ntsuas kub, B{{3} } yuav tsum xaiv , C1-2, C6-7, D6-7, B11-12, C11-12, vim cov ntsiab lus no yog cov ntsiab lus kub raws sijhawm.
