Universal SYBR GREEN qPCR Premix (Ubururu)

Injangwe No: HCB5041B

Universal Blue qPCR Master mix (Irangi rishingiye) nigisubizo kibanziriza 2 × igihe nyacyo cyo kugereranya PCR amplification irangwa no kwiyumvamo ibintu byinshi kandi byihariye, ni ubururu mu ibara, kandi bifite ingaruka zo gushakisha icyitegererezo.Ibyingenzi bigize Taq ADN polymerase ikoresha antibody itangiye kugirango ibuze neza amplification idasanzwe bitewe na primer annealing mugihe cyo gutegura icyitegererezo.Muri icyo gihe, formula yongeramo ibintu biteza imbere kunoza imikorere ya PCR no kuringaniza amplifike ya gen hamwe nibintu bitandukanye bya GC (30 ~ 70%), kugirango PCR yuzuye ibone umubano mwiza wumurongo muburyo bwinshi. karere.Ibicuruzwa birimo irangi ryihariye rya ROX Passive Reference Irangi, rikoreshwa mubikoresho byinshi bya qPCR.Ntabwo ari ngombwa guhindura concentration ya ROX kubikoresho bitandukanye.Birakenewe gusa kongeramo primers na templates kugirango utegure sisitemu ya reaction yo kwongera.

Ibigize

Isi yose yubururu qPCR Master mix

Imiterere yo kubika

Ibicuruzwa byoherejwe hamwe nudupapuro twa barafu kandi birashobora kubikwa kuri -25 ℃ ~ -15 ℃ mumezi 18.Birakenewe kwirinda imirasire yumucyo mugihe ubitse cyangwa utegura sisitemu yo kubyitwaramo.

Ibisobanuro

Amabwiriza

Sisitemu yo Kwitabira

[Icyitonderwa]: Kuvanga neza mbere yo gukoresha kugirango wirinde ibibyimba byinshi bitanyeganyega cyane.

a) Kwibanda kwa Primer: Kwibanda kwa primer kwanyuma ni 0.2μmol / L, kandi birashobora no guhinduka hagati ya 0.1 na 1.0μmol / L nkuko bikwiye.

b) Kwibandaho icyitegererezo: Niba inyandikorugero idakemuwe cDNA yibisubizo, ingano yakoreshejwe ntigomba kurenza 1/10 cyubunini bwa reaction ya qPCR.

c) Kwerekana icyitegererezo: Birasabwa kugabanya igisubizo cya cDNA inshuro 5-10.Umubare ntarengwa wicyitegererezo wongeyeho nibyiza mugihe Ct agaciro kabonetse nukwiyongera ni 20-30 cycle.

d) Sisitemu yo kubyitwaramo: Birasabwa gukoresha 20μL cyangwa 50μL kugirango tumenye neza kandi bisubirwemo intego yo kongera gene.

e) Gutegura sisitemu: Nyamuneka witegure mu ntebe isukuye cyane kandi ukoreshe inama nigituba kitagira ibisigisigi bya nuclease;birasabwa gukoresha inama hamwe na filteri ya karitsiye.Irinde kwanduza umusaraba no kwanduza aerosol.

2.Gahunda yo kubyitwaramo

Porogaramu isanzwe

Gahunda yihuse

Porogaramu

a) Ubushyuhe hamwe nigihe: Nyamuneka uhindure ukurikije uburebure bwa primer na gene intego.

b) Kugura ibimenyetso bya Fluorescence (★): Nyamuneka shiraho uburyo bwo kugerageza ukurikije ibisabwa mumabwiriza yo gukoresha igikoresho.

c) Gushonga umurongo: Porogaramu isanzwe igikoresho irashobora gukoreshwa mubisanzwe.

3. Isesengura ry'ibisubizo 

Nibura byibuze bitatu byigana bisabwa kugirango habeho igeragezwa ryinshi.Nyuma yo kubyitwaramo, amplification curve hamwe no gushonga umurongo bigomba kwemezwa.

3.1 Amplification curve:

Igipimo gisanzwe cyo kwongera umurongo ni S-shusho.Isesengura ryinshi nukuri iyo Ct agaciro kagabanutse hagati ya 20 na 30. Niba Ct agaciro kari munsi ya 10, birakenewe ko uhindura inyandikorugero hanyuma ukongera gukora ikizamini.Iyo Ct agaciro kari hagati ya 30-35, birakenewe kongera inyandikorugero yibanze cyangwa ingano ya sisitemu yogukora, kugirango tunoze imikorere ya amplification kandi tumenye neza ko isesengura ryibisubizo.Iyo agaciro ka Ct karenze 35, ibisubizo byikizamini ntibishobora gusesengura umubare wa gene, ariko birashobora gukoreshwa mubisesengura byujuje ubuziranenge.

3.2 Gushonga umurongo:

Impinga imwe yo gushonga umurongo yerekana ko reaction yihariye ari nziza kandi isesengura ryinshi rishobora gukorwa;niba gushonga umurongo byerekana impinga ebyiri cyangwa nyinshi, isesengura ryinshi ntirishobora gukorwa.Gushonga umurongo byerekana impinga ebyiri, kandi birakenewe kumenya niba impinga idateganijwe ari primer dimer cyangwa idasanzwe idasanzwe na ADN agarose gel electrophorei.Niba ari primer dimer, birasabwa kugabanya primer yibanze cyangwa kongera gushushanya primers hamwe nubushobozi buhanitse.Niba ari amplification idasanzwe, nyamuneka wongere ubushyuhe bwa annealing, cyangwa wongere ushushanye primers hamwe numwihariko.

Inyandiko

Nyamuneka wambare PPE ikenewe, ikote rya laboratoire hamwe na gants, kugirango ubuzima bwawe n'umutekano！

Ibicuruzwa nubushakashatsi bukoreshwa GUSA！