Iipolymerase chain reactions (PCR) yenye yeendlela ezaziwa ngokubanzi ezisetyenziswa kwiilabhoratri zesayensi yobomi.
Iipleyiti ze-PCR ziveliswa kwiiplastiki zodidi lokuqala ukuze zisebenze kakuhle kunye nohlalutyo lweesampulu okanye iziphumo eziqokelelweyo.
Zineendonga ezinqabileyo kunye ne-homogenous ukubonelela ngokutshintshela okuchanekileyo kwe-thermal.
Ukulungiselela usetyenziso lwexesha lokwenyani, icandelo lemizuzu ye-DNA okanye i-RNA livalelwe kwaye ligcinwe kwiipleyiti zePCR.
Iipleyiti zePCR zisebenza kakuhle kakhulu ekutywineni ubushushu kwaye zinqanda ukuhamba kobushushu ngokunjalo.
Nangona kunjalo, njengoko zisebenza kwaye zithembekile iipleyiti zePCR, impazamo kunye nokungachaneki kulula xa kusetyenzwa iisampulu.
Ke ngoko, ukuba unomdla wokufumana umgangatho olungileyo kunye nomgangatho ophezuluIipleyiti zePCR.Kukulungele ukuqhagamshelana nomenzi we-PCR othembekileyo. Ngale nto uqinisekile ngokufumana eyona ntengiso ingcono.
Nazi ezinye izilumkiso ekufuneka zilandelwe ukunqanda ukungcoliseka kwee-reagents okanye iisampulu kunye nokuthintela ukungachaneki ekungeneni kwiziphumo.
Ukucoca iiNdawo ezizingqongileyo
Iimpawu ezingafanelekanga okanye ezimbi zenzeka ngenxa yobukho bokungcola, okukwenza ukuba ungabaze iziphumo.
Ukungcola kunye nokungcola kwenzeka ngeendlela ezahlukeneyo ezifana ne-DNA enganxulumananga okanye izongezo zekhemikhali ezithi ekugqibeleni zinciphise ukusebenza kakuhle kunye nokusebenza kwempendulo.
Kukho iindlela ezininzi zokunciphisa kakhulu izinga lokungcoliseka kwepleyiti yePCR.
Ukusebenzisa iingcebiso zokucoca iintsholongwane yenye indlela eluncedo yokuthintela ukungcola ukuba kungene kwiisampulu zakho ngeepipettes.
Ukunikezela ngeseti yezixhobo ezicocekileyo, ezibandakanya i-pipettes kunye neeracks, ngokukodwa ukusetyenziswa kwe-PCR. Oku kuya kuqinisekisa ugqithiselo olungahoywayo lokungcola okanye ungcoliseko olujikeleze ilabhoratri.
Sebenzisa i-bleach, i-ethanol kwi-pipettes, ii-racks kunye neebhentshi ukucima ukungcola.
Beka isithuba esibekelweyo kuzo zonke izenzo zakho zePCR ukuze ucuthe ngakumbi ungcoliseko lwamasuntswana.
Sebenzisa iiglavu ezicocekileyo kwinyathelo ngalinye kwaye uzibuyisele rhoqo.
Iipleyiti zePCR
Hlola Ukugxila kunye nokucoceka kweSifanekiso.
Ukucoceka kwebhentshi kunye nezixhobo ezisetyenziselwa ukuhlalutya iisampuli kunye ne-PCR kufuneka zigcinwe. Kubalulekile ukuqinisekisa iqondo lokucoceka kweesampuli ngaphambi kokuhlalutya kunye nokucubungula.
Ngokubanzi, abahlalutyi bathathela ingqalelo inqanaba lokuxinana kunye nokucoceka kweesampulu zeDNA.
Zama umlinganiselo wokuthatha i-260nm/280nm kufuneka ungabi ngaphantsi kwe-1.8. Ngelixa i-wavelength elandelayo phakathi kwe-230nm kunye ne-320nm isetyenziselwa ukuchonga ukungcola.
Ngokomzekelo, iityuwa ze-chaotropic kunye nezinye iikhompawundi ze-organic zifunyenwe kwi-230nm yezinga lokufunxa. Ngelixa i-turbidity kwiisampulu ze-DNA nazo zichongiwe kwaye zingqinwe ngesantya sokufunxa esingama-320nm.
Kuphephe ukugcwala kakhulu iiPleti zePCR ngemveliso
Kangangoko kunqwenelwa ukuqhuba iimveliso ezininzi ngaxeshanye, kukhokelela ekungcolisekeni kweeplate zePCR.
Ukulayisha ngaphezulu iipleyiti zePCR ezinenkunkuma yeemveliso ezahlukeneyo kwaye kwenza kube nzima kakhulu ukuqinisekisa iisampulu.
Gcina iiRekhodi ze-Aliquot PCR Reagents
Imijikelo eqhubekayo yokukhenkcisa/yokunyibilika kunye nokusetyenziswa rhoqo kwe-aliquot kungonakalisa i-PCR reagents, ii-enzymes kunye nee-DNTP's ngokuphinda kuhlaziywe.
Soloko uzama ukujonga umlinganiselo we-aliquot osetyenziswayo ngelixa ulungiselela iisampulu eziza kuhlalutywa.
I-LIMS ekhethwayo ifaneleke ngakumbi ukulawula uluhlu kunye nesixa se-reagents kunye neesampuli ezifriziwe okanye zinyibilikisiwe.
Khetha Elona qondo lobushushu lilungileyo.
Ukuchola kunye nokusetyenziswa kweqondo lobushushu elingalunganga kuyenye indlela iziphumo zePCR eziqulathe impazamo.
Ngamanye amaxesha, impendulo ayihambi njengoko bekucwangcisiwe. Kunqweneleka ukunciphisa ubushushu be-annealing ukuze kube lula ukusabela okuyimpumelelo.
Nangona kunjalo, ukwehlisa iqondo lokushisa kwandisa amathuba obuxoki kunye nokubonakala kwe-primer dimers.
Kubalulekile ukuqinisekisa uhlalutyo lwegophe lokunyibilika xa usebenzisa iipleyiti ze-PCR njengophawu olulungileyo lokukhetha iqondo lobushushu elichanekileyo.
I-Primer design software inceda ngokuyila, ukubonelela ngeqondo lobushushu elichanekileyo le-annealing ngokunciphisa ngokuthe ngqo impazamo kwiipleyiti zePCR.
Ngaba ufuna ipleyiti yePCR ekumgangatho ophezulu?
Kwimeko apho ubucinga ukuba ungamfumana phi umenzi othembekileyo weIipleyiti zePCR. Ungaphindi ukhangele kuba ukwindawo elungileyo.
Ngobubelecofa apha ukuze uqhagamshelane nathikwiimveliso ezikumgangatho ophezulu kunye nenkonzo ngexabiso elingayi kwaphula ibhanki.
Ixesha lokuposa: Oct-30-2021