Imbeba Genotyping Kit

Injangwe No: HCR2021A

Iki gicuruzwa nigikoresho cyagenewe kumenyekana byihuse genotypes yimbeba, harimo gukuramo ADN ya sisitemu na sisitemu yo kongera PCR.Ibicuruzwa birashobora gukoreshwa muburyo bwa PCR byongerewe imbaraga kuva umurizo wimbeba, ugutwi, amano nizindi ngingo nyuma yo gutandukana byoroshye na Lysis Buffer na Proteinase k.Nta igogora ryijoro, gukuramo fenol-chloroform cyangwa kweza inkingi, byoroshye kandi bigabanya igihe cyo gukora ubushakashatsi.Igicuruzwa gikwiranye no kongera ibice bigenewe kugera kuri 2kb na multiplex PCR reaction hamwe na joriji 3 za primers.2 × Imbeba ya Tissue Direct PCR ivanze irimo ADN yakozwe na genetike polymerase, Mg²⁺, DNTPs hamwe na sisitemu ya buffer nziza kugirango itange uburyo bwiza bwo kongera imbaraga no kwihanganira inhibitor, kugirango reaction ya PCR ishobore gukorwa hongeweho inyandikorugero na primers no kuvugurura ibicuruzwa kuri 1 ×.Ibicuruzwa bya PCR byongerewe hamwe niki gicuruzwa bifite ishingiro rya "A" ku mpera ya 3 ′ kandi birashobora gukoreshwa muburyo bwa clon ya TA nyuma yo kwezwa.

Ibigize

Ububiko

Ibicuruzwa bigomba kubikwa kuri -25 ~ -15 ℃ kumyaka 2.Nyuma yo gushonga, Lysis Buffer irashobora kubikwa kuri 2 ~ 8 ℃ kugirango ikoreshwe mugihe gito, hanyuma ukavanga neza mugihe ukoresheje.

Gusaba

Iki gicuruzwa kibereye gusesengura imbeba ya knockout, gutahura transgenji, genotyping nibindi.

Ibiranga

1.Igikorwa cyoroshye: nta mpamvu yo gukuramo ADN genomic;

2.Porogaramu yagutse: ikwiranye no kwongerera imbaraga ibice bitandukanye byimbeba.

Amabwiriza

1.Kurekura ADN genomic

1) Gutegura lysate

Tissue lysate itegurwa ukurikije umubare wintangarugero yimbeba zigomba guterwa (lysate ya tissue igomba gutegurwa kurubuga ukurikije dosiye hanyuma ikavangwa neza kugirango ikoreshwe), kandi igipimo cya reagent zisabwa kurugero rumwe niki gikurikira:

2) Icyitegererezo cyo Gutegura na Lysis

Basabwe Gukoresha Tissue

Fata urugero rukwiye rw'icyitegererezo cy'imbeba mu miyoboro isukuye ya centrifuge, ongeramo 200μL ya tissue nshya lysate kuri buri cyuma cya centrifuge, vortex no kunyeganyega, hanyuma ushire kuri 55 ℃ kuri 30mins, hanyuma ushushe kuri 98 ℃ kuri 3mins.

3) Centrifugation

Shyira lysate neza na centrifuge kuri 12,000 rpm kuri 5mins.Indengakamere irashobora gukoreshwa nkicyitegererezo cyo kongera PCR.Niba inyandikorugero ikenewe mububiko, ohereza supernatant mubindi bikoresho bya sterile centrifuge hanyuma ubike kuri -20 ℃ ibyumweru 2.

2.Kwiyongera kwa PCR

Kuraho 2 × Imbeba ya Tissue Direct PCR ivanze -20 ℃ hanyuma ukonjesha ku rubura, vanga hejuru hanyuma utegure sisitemu ya reaction ya PCR ukurikije imbonerahamwe ikurikira (ikore ku rubura):

Icyitonderwa:

a) Amafaranga yongeweho ntagomba kurenza 1/10 cya sisitemu, kandi niba hiyongereyeho byinshi, amplification ya PCR irashobora guhagarikwa.

Basabwe PCR Ibisabwa

Icyitonderwa:

a) Ubushyuhe bwa Annealing: Hamwe na Tm agaciro ka primer, birasabwa gushyiraho ubushyuhe bwa annealing kuri Tm ntoya ya primer + 3 ~ 5 ℃.

Ibibazo rusange nibisubizo

1.Nta murongo ugenewe

1) Ibicuruzwa birenze urugero.Hitamo ingano ikwiye yicyitegererezo, mubisanzwe ntabwo irenze 1/10 cya sisitemu;

2) Ingano nini cyane.Koresha lysate inshuro 10 hanyuma wongere, cyangwa ugabanye ingano yicyitegererezo na re-lysis;

3) Ingero z'inyama ntabwo ari shyashya.Birasabwa gukoresha ingero nshya za tissue;

4) Ubwiza bwa primer.Koresha ADN genomic kugirango wongere imbaraga kugirango ugenzure ubuziranenge bwa primer kandi uhindure igishushanyo mbonera.

2.Kwiyongera kudasanzwe

1) Ubushyuhe bwa annealing buri hasi cyane kandi numubare wizunguruka ni mwinshi.Ongera ubushyuhe bwa annealing kandi ugabanye umubare wizunguruka;

2) Kwibanda ku cyitegererezo ni hejuru cyane.Mugabanye ingano yicyitegererezo cyangwa ugabanye inyandikorugero inshuro 10 nyuma yo kwongera;

3) Umwihariko wa primer umwihariko.Hindura igishushanyo mbonera.

Inyandiko

1.Kugirango wirinde kwanduzanya hagati yintangarugero, hagomba gutegurwa ibikoresho byinshi byo gutoranya, kandi hejuru yibikoresho birashobora gusukurwa hamwe na 2% sodium hypochlorite yumuti cyangwa isuku ya acide nucleic nyuma ya buri cyitegererezo niba bikenewe gukoreshwa inshuro nyinshi.

2.Birasabwa gukoresha imbeba nshya yimbeba, kandi ingano yicyitegererezo ntigomba kuba nini cyane kugirango wirinde kugira ingaruka kubisubizo bya amplification.

3.Lysis Buffer igomba kwirinda gukonjesha kenshi, kandi irashobora kubikwa kuri 2 ~ 8 ℃ kugirango ikoreshwe mugihe gito.Niba bibitswe ku bushyuhe buke, imvura irashobora kubaho, kandi igomba gushonga burundu mbere yo kuyikoresha.

4.PCR Ivanga igomba kwirinda gukonjesha kenshi, kandi irashobora kubikwa kuri 4 ℃ kugirango ikoreshwe mugihe gito.

5.Iki gicuruzwa nubushakashatsi bwubushakashatsi gusa kandi ntigomba gukoreshwa mugupima kwa muganga cyangwa kuvura.