Kugadziriswa kune chimwe chezvinhu zvePolymasease Chain Reaction (PCR) zvinogona kuchinja kunaka kwechigumisiro, kana nekuvandudza kana kuderedza chibereko chemuchero uye unhu kana kuburikidza nekuvandudza kugadzirisa kwezvakanaka uye kunzwisisika. Parameters iyo inokonzera mugumo unogona kunge iri yemuviri (kureva, kutonhora, nguva dzenguva) kana makemikari (kureva, template concentration, rudzi rwe enzyme rinoshandiswa). Izvi zvinotevera zvikonzero zvakakosha zvinoita kuti kubudirira kwePCR.
01 Clean Laboratory Mamiriro
Sezvo PCR ichikwanisa kuona imwe molecule yeDNA, zvinokosha kuti urambe wakagadzikana mu laboratori. Nguva dzose kupfeka magirazi matsva, kushandisa sterile glassware, tubes uye pipette mazano uye asina kugadzirisa zvigadziriswa, uye uchenese nzvimbo yebasa usati watanga basa.
Nguva dzose inosanganisira kutora maitiro, pasina template DNA uye isina enzyme, kuve nechokwadi kuti migumisiro inokonzerwa nekuwedzera kwekodzero yakakodzera.
Vanhu vazhinji vanovimbisa kuva nehumwe hwavo hwemhinduro dzisina kugoverwa kuita kuti matambudziko awedzere nyore uye pipettes dzakasarudzwa dzinowanzochengeterwa paCCR chete. DNase uye RNase-isina PCR tubes, mazano e-aerosol-resistant pipette uye kushanda mumoto unopisa uye chiedza cheVU zvakare nzira dzokuderedza zvinetso.
02 Chemical Components
- Iva nevhesi 18 kusvika ku24
- Usave nemadziro echipiri (ie, hairpin loops)
- Iva nekugoverana kwakakwana kweG / C neA / T vaviri
- Haisi kubatsirana kune mumwe nemumwe pamakumbo matatu-makumbo
- Iva nemhepo yakanyunguduka (Tm) inenge 5-10 degrees Celcius pasi pemvura inotonhora, iyo inowanzowanikwa pakati pe55 ne 65 degrees Celcius
Iyo Tm yezvikamu zviviri zvose inofanira kunge yakafanana nemigumisiro yakanaka.
03 Reaction Mix: Template uye Primers
Yakanakisisa primer concentrations iri pakati pe 0.1 ne 0.6 micromoles / L. Nhamba ye template yakasiyana zvichienderana nerudzi rweDNA (munhu, bhakitiriya, plasmid).
Zvine zvishoma zvakakwana zve template, kune dzimwe nzira dzokuvandudza mhinduro, sekuwedzera kwenhamba nhamba kana kushandiswa kwe "kutanga kutanga." Nguva dzose edzai maitiro matsva nemaitiro akanaka ekutora, kuve nechokwadi kuti vanoshanda pasi pemamiriro ako ekuenzanisa.
04 Reaction Mix: Basa reEnzyme
Kusunganidzwa kweMgCl2 mukusanganiswa kwekusangana kunogona kukonzera mhinduro yePCR uye kunogona kuita basa rinokosha mukuita zvakanyanya kufadza. Magnesium cation inogadzirisa zvinokonzerwa ne dNTPs kuti ibudise iyo substrate chaiyo iyo polymerase enzyme inoziva.
Kuenzana kwezviyero zvina zveDNTPs zvakare kunobatsira kuderedza polymerase kukanganisa kwenhamba. Zvimwe zvinokonzera zvakadai se betaine, BSA, detergents, DMSO, glycerol, uye pyrophosphatase zvinogonawo kukanganisa maitiro e enzyme kana kushanda zvakanaka.
05 Type of Thermocycler
Paunenge uchitenga mabhizimusi, funga mupfungwa kuti mamwe thermocyclers haana kunyatsoperera kupfuura vamwe pakuchengetedza chaiyo, kutoda kutonhora.
Iyi ndiyo imwe nzvimbo iyo inodhura isingabhadharwi munguva yakareba apo iwe unotarisana nekugadzirisa kushanda kana kushandisa zvinyorwa zvinodikanwa zvakasiyana-siyana zvekuwedzera kushandura.
Nzvimbo yakagadzirwa nemasvingo yakagadzirirwa maitiro anotarirwa kuti akwanise maratiro chaiwo e thermocycler uri kushandisa, uyewo anobatsira kugadzirisa kutaridzika kwemashure.
06 Purogiramu Zvitsva
Kuwedzerwa kwezvibereko kunogona kuwanikwa kuburikidza nekuwedzera nguva yekuwedzera pamusoro pemakumbo makumi maviri, kuti abhadhara enzyme shomanana kukurisa mamwe template. Kazhinji, pasi pemadhora makumi mana anokwana kukwidziridza pasi pe10 template molecules kumusana wakakura zvakakwana kuti uone pane ethidium bromide-stained agarose gel .