Sechitsva murabhoritari, harisi basa rakanaka kuongorora zvirimwa zvakanaka kubva muboka rezvirimwa zvine mwero wakaderera wekutendeuka.Chekutanga, DNA inofanirwa kutorwa kubva kunhamba huru yemasampuli imwe neimwe, uyezve majini ekunze anozoonekwa nePCR.Zvisinei, zvigumisiro zvinowanzovharwa uye mabhendi ane zvinhu zvishomanana nguva nenguva, asi hazvibviri kuziva kana pane zvisizvo zvekuonekwa kana kuonekwa kwenhema..Hazvibatsiri here kutarisana nemaitiro akadaro ekuedza nemigumisiro?Usanetseke, hama inokudzidzisa maitiro ekuvheneka kunze kwezvinomera zvakanaka zviri nyore uye nemazvo.

Danho 1

Dhizaini yekuona yekutanga

Sarudza iyo endogenous gene uye exogenous gene kuti ionekwe zvinoenderana nesample inoongororwa, uye sarudza mumiriri 100-500bp kutevedzana mujeni rekutanga dhizaini.Maprimers akanaka anogona kuve nechokwadi chechokwadi chemhedzisiro yekuonekwa uye kupfupisa nguva yekuona (ona appendix yezvinowanzo shandiswa zvinoonekwa zvekutanga).

Cherechedza: Iwo achangobva kugadzirwa maprimers anofanirwa kukwenenzvera mamiriro ekuita uye kuona huchokwadi, huchokwadi uye yekuona muganho wekuonekwa isati yaonekwa yakakura.

Danho rechipiri

Dhizaini yekuyedza protocol

Positive control: Shandisa iyo yakacheneswa DNA ine chidimbu chakatarwa setemplate kuona kana PCR reaction system uye mamiriro akajairwa.

Negative/blank control: Shandisa iyo DNA template kana ddH2O iyo isina chidimbu chakanangwa setemplate kuona kana paine kunobva tsvina muPCR system.

Yemukati mareferensi control: shandisa iyo primer/probe musanganiswa weiyo endogenous gene yesample kuti iongororwe kuti uone kana template ichigona kuonekwa nePCR.

Cherechedza:

Positive, negative/ blank controls and internal control controls zvinofanirwa kusetwa pabvunzo yega yega kuti iongorore huchokwadi hwezviyedzo zvabuda.

Kuedza kugadzirira

Usati washandisa, cherechedza kana mhinduro yacho yakasanganiswa zvakaenzana.Kana kunaya kwakawanikwa, inoda kunyungudutswa uye kusanganiswa maererano nemirairo isati yashandiswa.2 × PCR musanganiswa inoda kuvezwa pombi uye kusanganiswa kakawanda ne micropipette isati yashandiswa kudzivirira kusaenzana kweion kugovera.

Cherechedza:

Buritsa bhuku racho woriverenga nokungwarira, uye ita gadziriro usati waedza uchinyatsoenderana nezvinodiwa nebhuku racho.

Danho rechina

Gadzirira PCR reaction system

Zvinoenderana neprotocol yekuyedza, sanganisa maprimers, H2O, uye 2 × PCR musanganiswa zvakaenzana, centrifuge uye ugovagovera kune yega yega reaction chubhu.

Cherechedza:

Pakuyedzwa kukuru kana kwenguva refu, zvinokurudzirwa kushandisa PCR reaction system ine UNG enzyme, inogona kunyatso kudzivirira kusvibiswa kweaerosol kunokonzerwa nePCR zvigadzirwa.

Danho rechishanu

Wedzera reaction template

Uchishandisa Direct PCR tekinoroji, hapana chikonzero cheinonetesa nucleic acid yekuchenesa maitiro, iyo template yemuenzaniso inogona kugadzirwa mukati memaminitsi gumi, uye inoenderana PCR reaction system inogona kuwedzerwa.

Cherechedza:

Iyo nzira yecleavage ine zvirinani yekuona maitiro, uye chigadzirwa chakawanikwa chinogona kushandiswa kune akawanda ekuona maitiro.

5.1: Kuwedzera kwakananga kwemashizha

Zvinoenderana nehukuru hwemufananidzo uri mubhuku, cheka shizha matishu nehupamhi hwe2-3mm woiisa muPCR reaction system.

Cherechedza: Ita shuwa kuti zvidimbu zvemashizha zvakanyudzwa zvachose muPCR reaction solution, uye usawedzere mashizha akawandisa.

5.2: Nzira yekupatsanura mashizha

Cheka mashizha emashizha nehupamhi hwe 5-7mm uye uise mu centrifuge tube.Kana ukasarudza mashizha akakura, tapota dzivisa kushandisa matishu emutsinga mukuru weshizha.Pipette 50ul Buffer P1 lysate mu centrifuge chubhu kuti ive nechokwadi chekuti lysate inokwanisa kunyudza zvachose mashizha emashizha, kuiisa mumhepo inopisa kana bhati yesimbi, uye lyse pa 95 ° C kwemaminitsi mashanu kusvika gumi.

Wedzera 50ul Buffer P2 neutralization mhinduro uye sanganisa zvakanaka.Iyo lysate inoguma inogona kushandiswa se template uye yakawedzerwa kune PCR reaction system.

Cherechedza: Nhamba ye template iri pakati pe5-10% yePCR system, uye haifaniri kudarika 20% (somuenzaniso, mu 20μl PCR system, wedzera 1-2μl ye lysis solution, kwete kupfuura 4μl).

Danho rechitanhatu

PCR maitiro

Mushure mekuisa centrifuging iyo PCR reaction chubhu, inoiswa muPCR chiridzwa chekusimudzira.

Cherechedza:

Maitiro acho anoshandisa isina-yakacheneswa template yekukwidziridza, saka iyo nhamba yeamplification cycles ndeye 5-10 mamwe madhigirii pane kana uchishandisa yakacheneswa DNA template.

Danho rechinomwe

Kuonekwa kweElectrophoresis uye kuongororwa kwemhedzisiro

M: 100bp DNA Ladder

1\4: Yakanatswa DNA nzira

2\5: Yakananga PCR nzira

3\6: Kudzora kusina chinhu

QC:

Mhedzisiro yemhedzisiro yezvinodzora zvakasiyana-siyana zvakaiswa muchiyedzo zvinofanirwa kusangana nemamiriro anotevera.Kana zvisina kudaro, chikonzero chechinetso chinofanira kuongororwa, uye kuedza kunofanira kuitwa zvakare mushure mokunge dambudziko rabviswa.

Tafura 1. Zvakajairwa bvunzo mhinduro dzemapoka akasiyana ekudzora

* Kana iyo plasmid ichishandiswa seyakanaka kutonga, endogenous gene bvunzo mhedzisiro inogona kuve isina kunaka

Mutongo wemhedzisiro:

A. Mhedzisiro yejini yesample haina kunaka, zvichiratidza kuti DNA yakakodzera kuona PCR yakajairika haigone kutorwa kubva mumuenzaniso kana DNA yakabudiswa ine PCR reaction inhibitors, uye DNA inofanira kutorwa zvakare.

B. Mhedzisiro yejini inosara yesample yakanaka, uye mhedzisiro yebvunzo yejini yekunze haina kunaka, zvichiratidza kuti DNA yakakodzera kucherechedzwa kwePCR yakatorwa kubva mumuenzaniso, uye inogona kutongwa kuti XXX gene haina kuwanikwa mumuenzaniso.

C. Mhedzisiro yejini inosara yesample yakanaka, uye mhedzisiro yebvunzo yejini yekunze yakanaka, zvichiratidza kuti DNA yakakodzera kucherechedzwa kwePCR yakabudiswa kubva mumuenzaniso, uye sampuli yeDNA ine XXX gene.Miedzo yekusimbisa inogona kuitwa zvakare.

Nhanho yechisere

Dhizaini yekuona yekutanga

Mushure mekuedza, shandisa 2% sodium hypochlorite mhinduro uye 70% ethanol mhinduro kupukuta nzvimbo yekuedza kudzivirira kusvibiswa kwezvakatipoteredza.