Ukulungiswa kunoma yiyiphi inxalenye yempendulo yePolymerase Chain Reaction (PCR) ingakwazi ukuguqula umgangatho wesiphumo, mhlawumbi ngokuphucula okanye ukunciphisa isivuno semveliso kunye nemigangatho okanye ngokuphucula ukuphendulwa kwemeko kunye nobuzwe. Iiparameters ezichaphazela umphumo wokuphela unokuba ngumzimba (oko kukuthi, ukushisa, ixesha lokujikeleza) okanye imichiza (oko kukuthi, i-template concentration, uhlobo lwe-enzyme elisetyenziswayo). Ezi zilandelayo zizinto eziphambili ezibalulekileyo ezichaphazela impumelelo ye-PCR.
01 Iingxaki zeLebhu yeeLayil
Ekubeni i- PCR inokukwazi ukubona i-molecule enye ye-DNA, kubalulekile ukugcina imeko ecacileyo kwibhubhoratri. Njalo ugqoke iiglavu ezitsha, sebenzisa i-glassware engenamanzi, iipilisi kunye neengcebiso zepipette kunye nezisombululo ezinobumba, kwaye uhlambulule indawo yomsebenzi ngaphambi kokuba uqale umsebenzi.
Njalo uquka ukuphendula, ngaphandle kwe-DNA ye template kwaye ngaphandle kwe-enzyme, ukuqinisekisa ukuba iziphumo zibangelwa ukukhulisa isampuli esifanelekileyo.
Uninzi lwabantu luqinisekisa ukuba nesisombululo sazo esabelana ngazo ukuze senze inkathazo ngokulula kwaye iipipettes ezikhethiweyo zihlala zibeka eceleni kwi-PCR kuphela. I-DNase ne-RNase-free of tuberculosis PCR, iingcebiso ze-aerosol ezingamelana ne-pipette kunye nokusebenza kwindawo yokuvutha komlilo kunye nokukhanya kwe-UV ziindlela zokunciphisa iingxaki.
02 IiComputer Components
- Unamaziko angama-18 ukuya kuma-24
- Awunaso isakhiwo sesibini (okt, i-loop hairpin)
- Ukwabiwa ngokulinganayo kwee-G / C kunye ne-A / T ezimbini
- Azincedisi omnye komnye kwii-3-foot-ends
- Ukushisa ukushisa (Tm) malunga ne-5-10 degrees Celcius ngaphantsi kweqondo lokushushu, okuqhelekileyo phakathi kwama-55 no-65 degrees Celcius
I-Tm yezibini zombini kufuneka zifanane neziphumo ezilungileyo.
03 Umxube wokuphendula: I-Template kunye neeNqununu
Iimpawu eziphambili ze-primer ziphakathi kwe-0.1 ne-0.6 micromoles / L. Isixa sethemplate sihluka ngohlobo lwe-DNA (umntu, ibhakteria, iplasmid).
Ngeemali eziphantsi kakhulu zeethemplate, zikho ezinye izicwangciso zokuphucula iziphumo, njengamanani omjikelezo okwandisiweyo okanye ukusebenzisa "ukuqala okutshisa." Njalo uvavanye amavali amasha ngokuphendula okulungileyo, ukuqinisekisa ukuba basebenza phantsi kweemeko zakho zokulinga.
04 Umxube wokuphendula: Umsebenzi we-Enzyme
Ukuxinwa kwe-MgCl2 kumxube weempendulo kunokuchaphazela umphumo we-PCR kwaye unokudlala indima ebalulekileyo kwiimpendulo ezithandekayo. I-Magnesium cation iyenzela i-dNTPs i-soluble complexes ukuvelisa i-substrate yangempela ukuba i-enzyme ye-polymerase iyakwazi.
Inani elilinganayo lazo zonke i-dNTPs linceda ukunciphisa izinga lokuphosa iipolymase. Ezinye izongezo ezifana ne-betaine, i-BSA, i-detergents, i-DMSO, i-glycerol, ne-pyrophosphatase inokuchaphazela inkcazo ye-enzyme okanye ukusebenza kakuhle.
05 Uhlobo lwe-Thermocycler
Xa uthengisa izixhobo zelabhoratri, khumbula ukuba ezinye i-thermocyclers zichanekile kunabanye ukugcina amaqondo afanelekileyo, afudumeleyo.
Le yindawo enye ekuhlawulekayo ayiyi kuhlawula ngokukhawuleza xa ujongene nokuphendula ngokugqithiseleyo okanye usebenzisa iimbumba ezifuna uluhlu oluncinci lobushushu bokususa.
Iibhubhu zokuphendula ezinamacinci ezenzelwe ukulungelelanisa uphawu oluthile lwe-thermocycler oyisebenzisayo, kwakhona lunceda ukwandisa izinga lokushisa.
06 Izicwangciso zeeKrikhi
Ukuvelisa isivuno kungenziwa ngokunyusa ixesha lokunyusa malunga neesigidi ezili-20, ukuhlawulela i-enzyme encinci ukukhulisa ithemplate engaphezulu. Ngokuqhelekileyo, imijikelezo engaphantsi kwama-40 ngokwaneleyo ukukhulisa i-molecule ye-template engaphantsi kwe-10 ibe yinkxalabo enkulu ngokwaneleyo ukujonga kwi-ethidium bromide-stained agarose gel .