Kukhazikika kwa njira kumaphatikizapo kukhathamiritsa kwawo ndi kukhazikitsidwa kotsatira ndi kugwirizanitsa, kulola kuchita bwino kwa nthawi yayitali - osadalira wogwiritsa ntchito.Kukhazikika kumatsimikizira zotsatira zapamwamba, komanso kuberekana kwawo komanso kufananiza.
Cholinga cha (chachikale) PCR ndikutulutsa zotsatira zodalirika komanso zobwereketsa.Kwa mapulogalamu ena, zokolola zaMtengo wa PCRndizofunikanso.Pamachitidwe awa, chisamaliro chiyenera kuchitidwa kuti zitsanzo sizisokonezedwa komanso kuti mayendedwe a PCR azikhala okhazikika.Makamaka, izi zikutanthawuza kuchepetsa kuyambitsidwa kwa zoyipitsidwa zomwe zitha kubweretsa zotsatira zabodza kapena zabodza kapenanso kulepheretsa machitidwe a PCR.Kuphatikiza apo, zomwe zimachitikira ziyenera kukhala zofanana momwe zingathere kwa munthu aliyense payekhapayekha komanso kusamutsidwa kuzinthu zotsatila (zanjira yomweyo).Izi zikutanthawuza kupangidwa kwa machitidwe komanso mtundu wa kutentha kwa woyendetsa.Zolakwa za ogwiritsa ntchito, ndithudi, ziyenera kupewedwa momwe zingathere.
Pansipa, tiwonetsa zovuta zomwe timakumana nazo panthawi yokonzekera komanso nthawi yonse ya PCR - ndi njira zothetsera mavuto omwe alipo pokhudzana ndi zida ndi zinthu zomwe zimagwiritsidwa ntchito poyimilira ma PCR.
Kukonzekera zochita
Kugawa kwazinthu zomwe zimakhudzidwa ndi PCR-zotengera, kapena mbale, motsatana, zimakhala ndi zovuta zingapo zomwe ziyenera kuthetsedwa:
Zomwe zimachitika
Yeniyeni ndi yeniyeni mlingo wa munthu zigawo zikuluzikulu ndi wofunika kwambiri polimbana kwambiri zofanana anachita zinthu zotheka.Kuphatikiza pa njira yabwino yopangira mapaipi, kusankha chida choyenera ndikofunikira.Zosakaniza za PCR master-mix nthawi zambiri zimakhala ndi zinthu zomwe zimawonjezera kukhuthala kapena kupanga thovu.Pa ndondomeko ya pipetting, izi zimabweretsa kunyowa kwakukulu kwamalangizo a pipette, motero kuchepetsa kulondola kwa pipetting.Kugwiritsiridwa ntchito kwa machitidwe operekera mwachindunji kapena nsonga zina za pipette zomwe sizimanyowetsa pang'onopang'ono zimatha kupititsa patsogolo kulondola ndi kulondola kwa ndondomeko ya pipetting.
Kuipitsidwa
Panthawi yoperekera, ma aerosols amapangidwa, omwe, ngati aloledwa kufika mkati mwa pipette, akhoza kuwononga chitsanzo china panthawi yotsatira ya pipetting.Izi zitha kupewedwa pogwiritsa ntchito nsonga zosefera kapena njira zosinthira mwachindunji.
Consumables mongamalangizo, zombo ndi mbale zomwe zimagwiritsidwa ntchito mu PCR siziyenera kukhala ndi zinthu zomwe zimasokoneza chitsanzo kapena chinyengo chotsatira.Izi zikuphatikizapo DNA, DNases, RNases ndi PCR inhibitors, komanso zigawo zomwe zingathe kuchotsedwa kuchokera kuzinthu zomwe zimapangidwira - zinthu zomwe zimatchedwa leachables.
Zolakwika za ogwiritsa
Zitsanzo zambiri zikakonzedwa, m'pamenenso pali chiopsezo chokwera.Zitha kuchitika mosavuta kuti sampuli imalowetsedwa mu chotengera cholakwika kapena pachitsime cholakwika.Chiwopsezochi chikhoza kuchepetsedwa kwambiri pozindikirika mosavuta pazitsime.Kupyolera mu makina opangira masitepe, "chinthu chaumunthu", mwachitsanzo, zolakwika ndi zosiyana zokhudzana ndi ogwiritsa ntchito, zimachepetsedwa, motero zimawonjezera kuberekana, makamaka pazochitika zazing'ono.Izi zimafuna mbale zokhazikika zokwanira kuti zigwiritsidwe ntchito pogwirira ntchito.Ma barcode omwe aphatikizidwa amawonjezera kuwerengeka kwa makina, zomwe zimathandizira kutsata zitsanzo munthawi yonseyi.
Kukonzekera kwa thermocycler
Kupanga chida kumatha kukhala kowononga nthawi komanso kosavutikira.Zinthu zosiyanasiyana za PCR zotenthetsera zimagwirira ntchito limodzi kuti izi zisakhale zophweka, ndipo, koposa zonse, kuti zikhale zotetezeka:
Kugwiritsa ntchito kosavuta komanso chitsogozo chabwino cha ogwiritsa ntchito ndizo maziko a pulogalamu yabwino.Kumanga pamaziko awa, kasamalidwe ka ogwiritsa ntchito otetezedwa ndi mawu achinsinsi kuletsa mapulogalamu ake kuti asasinthidwe ndi ogwiritsa ntchito ena.Ngati ma cyclers angapo (amtundu womwewo) akugwiritsidwa ntchito, ndizothandiza ngati pulogalamu ingasamutsidwe kuchokera ku chida chimodzi kupita ku china kudzera pa USB kapena kulumikizana.Mapulogalamu apakompyuta amathandizira kasamalidwe kapakati komanso kotetezeka kwa mapulogalamu, ufulu wa ogwiritsa ntchito ndi zolemba pakompyuta.
Kuchita kwa PCR
Panthawi yothamanga, DNA imakulitsidwa muchombo chochitira, pomwe chitsanzo chilichonse chiyenera kuchitidwa mofanana, chosagwirizana.Zotsatirazi ndizogwirizana ndi ndondomekoyi:
Kuwongolera kutentha
Kulondola kwabwino kwambiri pakuwongolera kutentha ndi homogeneity ya chipika cha cycler ndi maziko a ngakhale kutentha kwa zitsanzo zonse.Ubwino wapamwamba wa zinthu zotenthetsera ndi zoziziritsa kukhosi (zinthu za peltier), komanso momwe izi zimalumikizirana ndi chipika, ndizosankha zomwe zidzatsimikizire kuopsa kwa kusiyana kwa kutentha komwe kumadziwika kuti "m'mphepete"
Evaporation
The woipa wa munthu anachita zigawo zikuluzikulu sayenera kusintha m'kupita kwa anachita chifukwa evaporation.Apo ayi, ndizotheka kuti zochepa kwambiriMtengo wa PCRakhoza kupangidwa, kapena ayi konse.Choncho ndikofunika kuchepetsa kutuluka kwa nthunzi poonetsetsa kuti chisindikizo chili chotetezeka.Pankhaniyi, chivindikiro chotenthetsera cha thermocycler ndi chisindikizo cha chotengeracho chimagwira ntchito limodzi.Zosankha zosindikiza zosiyanasiyana zilipoMa mbale a PCR (ulalo: Nkhani yosindikiza), momwe chisindikizo chabwino kwambiri chimapezedwa mwa kusindikiza kutentha.Kutseka kwina kungakhalenso koyenera, bola ngati kulumikizidwa kwa chivindikiro cha cycler kungasinthidwe ku chisindikizo chosankhidwa.
Kuyimitsidwa kwa ndondomeko kuli m'malo kuti muteteze zotsatira zolondola komanso zobwerezabwereza pakapita nthawi.Izi zikuphatikizapo kukonza nthawi zonse zipangizo kuti zitsimikizire kuti nthawi zonse zimagwira ntchito bwino.Zogulitsa zonse ziyenera kukhala zapamwamba kwambiri pamagawo onse opangidwa, ndipo kupezeka kwake kodalirika kuyenera kutsimikiziridwa.
Nthawi yotumiza: Nov-29-2022