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Njengomtsha kwilabhoratri, ayingomsebenzi ulungileyo ukuhluza izityalo eziqinisekileyo ukusuka kwiqela lezityalo ezinezinga eliphantsi loguqulo.Okokuqala, i-DNA kufuneka ikhutshwe kwinani elikhulu leisampulu nganye nganye, kwaye emva koko iijini zangaphandle ziya kubonwa yi-PCR.Nangona kunjalo, iziphumo zihlala zingenanto kunye neebhendi ezinezinto ezimbalwa ngamaxesha athile, kodwa akunakwenzeka ukumisela ukuba ngaba kukho ubhaqo oluphosiweyo okanye ukufunyanwa kobuxoki..Ngaba akuncedi kakhulu ukujongana nenkqubo yovavanyo kunye neziphumo?Sukuba nexhala, umzalwana ukufundisa indlela yokuhluza izityalo ze-transgenic positive ngokulula nangokuchanekileyo.

Inyathelo loku-1

Iiprayimari zokubona ukuyila

Ngokukhawuleza1

Qinisekisa i-endogenous gene kunye ne-exogenous gene ukuba ibonwe ngokuhambelana nesampuli ukuba ihlolwe, kwaye ukhethe ummeli we-100-500bp ulandelelwano kwi-gene yoyilo lwe-primer.Ii-primers ezilungileyo zinokuqinisekisa ukuchaneka kweziphumo zokufumanisa kunye nokunciphisa ixesha lokufumanisa (jonga i-appendix kwii-primers ezisetyenziswa ngokuqhelekileyo).

Isaziso:Iiprimers ezisandula ukuyilwa kufuneka zongeze iimeko zokusabela kwaye ziqinisekise ukuchaneka, ukuchaneka kunye nomda wokubhaqa wobhaqo phambi kokubhaqwa komgangatho omkhulu.

Inyathelo lesi-2

Yila iprotocol yokulinga

Rapid2

Ulawulo oluhle: Sebenzisa i-DNA ecocekileyo equlethe iqhekeza ekujoliswe kulo njenge template ukujonga ukuba inkqubo ye-PCR yokusabela kunye neemeko ziqhelekileyo.

Ulawulo olubi / olungenanto: Sebenzisa i-template ye-DNA okanye i-ddH2O engenalo iqhekeza ekujoliswe kulo njenge template ukubona ukuba kukho umthombo wokungcola kwinkqubo ye-PCR.

Ulawulo lwangaphakathi lwereferensi: sebenzisa i-primer/probe indibaniselwano ye-endogenous gene yesampulu ukuze ivavanywe ukuvavanya ukuba itemplate inokufunyanwa yi-PCR.

Isaziso:

Ulawulo oluhle, olubi / olungenanto kunye nolawulo lwangaphakathi kufuneka lubekwe kuvavanyo ngalunye ukuvavanya ukunyaniseka kweziphumo zovavanyo.

Ukulungiselela ukulinga

Rapid3

Ngaphambi kokusetyenziswa, jonga ukuba isisombululo sixutywe ngokulinganayo.Ukuba imvula ifunyenwe, kufuneka inyibilikiswe kwaye ixutywe ngokwemiyalelo phambi kokusetyenziswa.I-2 × i-PCR yokuxuba kufuneka ifakwe ipayipi kwaye ixutywe ngokuphindaphindiweyo kunye ne-micropipette ngaphambi kokusetyenziswa ukuphepha ukusabalalisa i-ion engalinganiyo.

Isaziso:

Khupha imanyuwali kwaye uyifunde ngocoselelo, kwaye wenze amalungiselelo phambi kovavanyo ngokungqongqo ngokungqinelana neemfuno zemanyuwali.

Inyathelo lesi-4

Lungisa inkqubo ye-PCR yokusabela

Rapid4

Ngokweprothokholi yovavanyo, xuba iiprimers, i-H2O, kunye ne-2 × PCR xuba ngokulinganayo, i-centrifuge kwaye usasaze kuyo ityhubhu yokusabela nganye.

Isaziso:

Kuvavanyo olukhulu okanye olude, kucetyiswa ukuba kusetyenziswe inkqubo ye-PCR yokusabela equkethe i-UNG enzyme, enokuthintela ngokufanelekileyo ukungcoliseka kwe-aerosol okubangelwa yimveliso ye-PCR.

Inyathelo lesi-5

Yongeza itemplate yokusabela

Rapid5

Ukusebenzisa iteknoloji ye-PCR ethe ngqo, akukho mfuneko yenkqubo yokuhlanjululwa kwe-nucleic acid edinayo, i-template yesampuli inokulungiswa kwimizuzu eyi-10, kwaye inkqubo yokusabela kwe-PCR ehambelanayo inokongezwa.

Isaziso:

Indlela ye-cleavage inesiphumo esingcono sokufumanisa, kwaye imveliso efunyenweyo ingasetyenziselwa iimpendulo ezininzi zokubona.

Rapid6

5.1: Ukwandiswa ngokuthe ngqo kwamagqabi

Ngokobukhulu bomfanekiso kwincwadana, nquma isicatshulwa seqabunga kunye nobubanzi be-2-3mm kwaye uyibeke kwinkqubo yokusabela kwe-PCR.

Qaphela: Qinisekisa ukuba amaqhekeza amagqabi antywiliselwe ngokupheleleyo kwisisombululo sokusabela kwe-PCR, kwaye ungafaki izicubu zamagqabi ezigqithisileyo.

5.2: Indlela yokwahlula amagqabi

Sika izicubu zeqabunga kunye nobubanzi be-5-7mm kwaye uyibeke kwi-tube ye-centrifuge.Ukuba ukhetha amagqabi aqolileyo, nceda ugweme ukusebenzisa izicubu zomthambo ophambili wegqabi.I-Pipette 50ul Buffer P1 i-lysate kwi-tube ye-centrifuge ukuqinisekisa ukuba i-lysate inokuntywilisela ngokupheleleyo i-tissue yeqabunga, ibeke kwi-cycler ye-thermal okanye i-metal bath, kunye ne-lyse kwi-95 ° C kwi-5-10 imizuzu.

Rapid7

Yongeza i-50ul Buffer P2 isisombululo sokungathathi hlangothi kwaye udibanise kakuhle.I-lysate ephumayo ingasetyenziswa njenge template kwaye yongezwe kwi-PCR reaction system.

Qaphela: Isixa se-template siphakathi kwe-5-10% yenkqubo ye-PCR, kwaye akufanele idlule i-20% (umzekelo, kwi-20μl inkqubo ye-PCR, yongeza i-1-2μl yesisombululo se-lysis, ingabi ngaphezu kwe-4μl).

Inyathelo lesi-6

Ukusabela kwePCR

Ngokukhawuleza8

Emva kwe-centrifuging ityhubhu yokusabela kwe-PCR, ibekwe kwisixhobo se-PCR sokukhulisa.

Isaziso:

Ukusabela kusebenzisa itemplate engahlanjululwanga yokukhulisa, ngoko ke inani lemijikelezo yokukhulisa i-5-10 imijikelezo engaphezulu xa usebenzisa i-template ye-DNA ecocekileyo.

Inyathelo lesi-7

Ukufunyanwa kwe-Electrophoresis kunye nohlalutyo lweziphumo

Rapid9

M: 100bp DNA Ileli

I-1\4: Indlela ye-DNA ecocekileyo

2\5: Indlela ye-PCR ethe ngqo

3\6: Ulawulo olungenanto

QC:

Iziphumo zovavanyo lweendlela ezahlukeneyo zolawulo olusetwe kuvavanyo kufuneka zihlangabezane nezi meko zilandelayo.Ngaphandle koko, imbangela yengxaki kufuneka ihlalutywe, kwaye uvavanyo kufuneka lwenziwe kwakhona emva kokuba ingxaki isuswe.

Uluhlu 1. Iziphumo zovavanyo eziqhelekileyo zamaqela ahlukeneyo olawulo

*Xa iplasmid isetyenziswa njengolawulo olulungileyo, iziphumo zovavanyo lwemfuza endogenous zinokuba zimbi

Isigwebo sesiphumo:

A. Isiphumo sovavanyo lwe-endogenous gene yesampulu sibi, esibonisa ukuba i-DNA ifanelekile ukufunyanwa kwe-PCR eqhelekileyo ayikwazi ukukhutshwa kwisampuli okanye i-DNA ekhutshiweyo iqulethe i-PCR reaction inhibitors, kwaye i-DNA kufuneka ikhutshwe kwakhona.

B. Iziphumo zovavanyo lwe-endogenous gene yesampulu zilungile, kwaye iziphumo zovavanyo lwe-exogenous gene zibi, ebonisa ukuba i-DNA ilungele ukufunyaniswa kwe-PCR eqhelekileyo ikhutshwe kwisampulu, kwaye inokugwetywa ukuba i-XXX gene ayibonwanga kwisampulu.

C. Iziphumo zovavanyo lwe-endogenous gene yesampulu zilungile, kwaye umphumo wovavanyo we-exogenous gene ulungile, ebonisa ukuba i-DNA efanelekileyo yokufunyanwa kwe-PCR eqhelekileyo ikhutshwe kwisampuli, kwaye isampuli ye-DNA iqulethe i-XXX gene.Imifuniselo yokuqinisekisa inokuqhutywa ngakumbi.

Inyathelo lesi-8

Iiprayimari zokubona ukuyila

Rapid10

Emva kovavanyo, sebenzisa i-2% yesisombululo se-sodium hypochlorite kunye ne-70% yesisombululo se-ethanol ukusula indawo yovavanyo ukukhusela ukungcoliseka kwendalo.


Ixesha lokuposa: Sep-08-2021