Isishwankathelo
Ukuchongwa ngokukhawuleza kwezityalo eziguqukayo
Umbhalo/Tong Yucheng
Umsebenzi wokulinga/uHan Ying
Umhleli/Wen Youjun
Amagama/1600+
Ixesha lokufunda elicetyiswayo / imizuzu eyi-8-10
Ukuchongwa ngokukhawuleza kwezityalo eziguqukayo
Njengomntu osandul' ukufika elabhoratri, ayingomsebenzi ulungileyo ukuhluza izityalo eziqinisekileyo ukusuka kwiqela lezityalo ezinezinga eliphantsi lokuguqulwa.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
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).
Phawula:
Iiprimers ezisandula ukuyilwa kufuneka zongeze iimeko zokusabela kwaye ziqinisekise ukuchaneka, ukuchaneka, kunye nomda wokufumanisa ngaphambi kokwenza ubhaqo olukhulu.
Inyathelo lesi-2:Phuhlisa iprothokholi yokulinga
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 itemplate yeDNA okanye iddH2O engaqulathanga iqhekeza ekujoliswe kulo njenge template ukubona ukuba kukho umthombo wongcoliseko kwinkqubo yePCR.
Ulawulo lwangaphakathi lwereferensi: sebenzisa i-primer/probe indibaniselwano ye-endogenous gene yesampulu ukuze ivavanywe ukuvavanya ukuba itemplate inokufunyanwa yi-PCR.
Phawula:
Ulawulo oluhle, olubi / olungenanto kunye nolawulo lwangaphakathi kufuneka lubekwe kuvavanyo ngalunye ukuvavanya ukunyaniseka kweziphumo zovavanyo.
Inyathelo lesi-3: Ukulungiselela ukulinga
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.
Phawula:
Thatha imiyalelo kwaye uyifunde ngononophelo, kwaye wenze amalungiselelo phambi kovavanyo ngokuhambelana ngqongqo nemiyalelo.
Inyathelo 4: Lungiselela inkqubo yokusabela kwePCR
Ngokweprothokholi yovavanyo, xuba iiprimers, H2O, 2 × PCR mix, centrifuge kwaye usasaze kubo ityhubhu yokusabela nganye.
Phawula:
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 5: Yongeza itemplate yokusabela
Ukusebenzisa itekhnoloji ye-PCR ethe ngqo, akukho mfuneko yenkqubo yokucoca i-nucleic acid edinayo.Ithemplethi yesampula inokulungiswa ngaphakathi kwemizuzu eyi-10 kwaye yongezwe kwinkqubo yokusabela ye-PCR ehambelanayo.
Phawula:
Indlela yeLysis inesiphumo esingcono sokufumanisa, kwaye imveliso efunyenweyo ingasetyenziselwa ukuphendulwa kokubona okuninzi.
5.1: I-PCR ethe ngqo yamagqabi
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 yokucoca 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.
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 kufuneka sibe phakathi kwe-5-10% yenkqubo ye-PCR, kwaye akufanele idlule i-20% (umzekelo, kwi-20μl inkqubo ye-PCR, yongeza i-1-2μl ye-lysis buffer, ingabi ngaphezu kwe-4μl).
Inyathelo lesi-6:Ukusabela kwePCR
Emva kokwenza i-centrifuging i-PCR reaction tube, yibeke kwisixhobo se-PCR sokukhulisa.
Phawula:
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
M:100bp Ileli yeDNA
I-1\4: Indlela ye-DNA ecocekileyo
2\5: Indlela ye-PCR ethe ngqo
3\6: Ulawulo olungenanto
Ulawulo lwemeko:
Iziphumo zovavanyo lweendlela ezahlukeneyo zolawulo ezibekwe 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. Isiphumo sovavanyo lwe-endogenous gene yesampulu silungile, kwaye iziphumo zovavanyo lwe-exogenous gene zibi, ebonisa ukuba i-DNA ilungele ukufunyaniswa kwe-PCR eqhelekileyo ikhutshwe kwisampulu, kwaye inokugwetywa ukuba i-XXX yemfuza ayibonwanga kwisampulu.
C. Iziphumo zovavanyo lwe-endogenous gene yesampulu zilungile, kwaye iziphumo zovavanyo lwe-exogenous gene zilungile, 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: Yila iiprimer zokubona
Emva kovavanyo, sebenzisa i-2% yesisombululo se-sodium hypochlorite kunye ne-70% isisombululo se-ethanol ukusula indawo yokuhlola ukukhusela ukungcoliseka kwendalo.
Isihlomelo
Itheyibhile 2. Iiprimers ezisetyenziswa ngokuqhelekileyo kwi-PCR jikelele yokubona izityalo eziguqulwe ngofuzo
Uxwebhu lweReferensi:
I-SN / T 1202-2010, Indlela yokufumanisa i-PCR esemgangathweni yezithako zezityalo eziguqulwe ngofuzo ekudleni.
Isibhengezo soMphathiswa wezoLimo 1485-5-2010, Uvavanyo lwezithako zezityalo eziguqulwe ngokwemfuza kunye nemveliso yazo-irayisi M12 kunye nezinto eziphuma kuyo.
Ixesha lokuposa: Jun-09-2021
