Mouse Genotyping Kit

Nomoro ea katse: HCR2021A

Sehlahisoa sena ke khiti e etselitsoeng ho tsebahatsa ka potlako mefuta ea litoeba, ho kenyeletsoa DNA crude extraction le PCR amplification system.Sehlahisoa se ka sebelisoa bakeng sa ho hōlisa PCR ka ho toba ho tloha mohatleng oa mouse, tsebe, menoana ea maoto le lisele tse ling ka mor'a hore ho be bonolo ho phunyeha ka Lysis Buffer le Proteinase k.Ha ho na tšilo ea lijo ka bosiu bo le bong, ho ntšoa ha phenol-chloroform kapa tlhoekiso ea likholomo, e bonolo ebile e khutsufatsa nako ea liteko.Sehlahisoa se loketse ho holisa likhechana tse shebiloeng ho fihla ho 2kb le multiplex PCR ka li-primers tse fihlang ho tse 3.2 × Mouse Tissue Direct PCR Mix e na le DNA polymerase e entsoeng ka liphatsa tsa lefutso, Mg.²⁺, dNTPs le sistimi e ntlafalitsoeng ea buffer ho fana ka katleho e phahameng ea amplification le mamello ea inhibitor, e le hore karabelo ea PCR e ka etsoa ka ho eketsa template le li-primers le ho khutlisetsa sehlahisoa ho 1 ×.Sehlahisoa sa PCR se matlafalitsoeng ka sehlahisoa sena se na le setsi se hlaheletseng sa "A" qetellong ea 3 'me se ka sebelisoa ka ho toba bakeng sa TA cloning ka mor'a ho hloekisoa.

Likaroloana

Maemo a polokelo

Lihlahisoa li lokela ho bolokoa ho -25 ~ -15 ℃ bakeng sa lilemo tse 2.Kamora ho qhibiliha, Lysis Buffer e ka bolokoa ho 2 ~ 8 ℃ bakeng sa ts'ebeliso ea nako e khuts'oane, 'me e kopane hantle ha e sebelisoa.

Kopo

Sehlahisoa sena se loketse tlhahlobo ea ho kokota ha mouse, tlhahlobo ea transgenic, genotyping joalo-joalo.

Likaroloana

1.Ts'ebetso e bonolo: ha ho hlokahale ho ntša DNA ea genomic;

2.Ts'ebeliso e pharaletseng: e loketse ho holisa ka kotloloho lithane tse fapaneng tsa toeba.

Litaelo

1.Ho lokolloa ha genomic DNA

1) Tokisetso ea lysate

Li-tissue lysate li lokisoa ho latela palo ea lisampole tsa toeba tse lokelang ho etsoa lysed (li-tissue lysate li lokela ho lokisoa sebakeng sa marang-rang ho latela litekanyetso le ho tsoakoa ka botlalo bakeng sa ts'ebeliso), mme karolo ea li-reagents tse hlokahalang bakeng sa sampole e le 'ngoe ke tse latelang:

2) Tokiso ea Mohlala le Lysis

Tšebeliso ea Tissue e khothalelitsoeng

Nka palo e loketseng ea lisampole tsa masela a toeba ka har'a li-tubes tse hloekileng tsa centrifuge, eketsa 200μL ea li-tissue tse ncha tsa lysate ho tube e 'ngoe le e' ngoe ea centrifuge, vortex le shake, ebe u kenya 55 ℃ bakeng sa 30mins, ebe u chesa ho 98 ℃ bakeng sa 3mins.

3) Centrifugation

Shake lysate hantle 'me u centrifuge ka 12,000 rpm bakeng sa 5mins.Supernatant e ka sebelisoa e le template bakeng sa ho holisa PCR.Haeba thempleite e hlokahala bakeng sa polokelo, fetisetsa supernatant ho e 'ngoe sterile centrifuge tube 'me u boloke ho -20 ℃ bakeng sa libeke tse 2.

2.Matlafatso ea PCR

Tlosa 2 × Mouse Tissue Direct PCR Mix ho tloha -20 ℃ 'me u qhibilihe holim'a leqhoa, kopanya ka holimo le ho lokisa mokhoa oa ho arabela oa PCR ho latela tafole e latelang (sebetsa holim'a leqhoa):

Hlokomela:

a) Chelete e ekelitsoeng ha ea lokela ho feta 1/10 ea sistimi, mme haeba e eketsoa haholo, ho holisa PCR ho ka thibeloa.

Lipehelo tsa PCR tse khothalelitsoeng

Hlokomela:

a) Mocheso oa ho kopanya: Ha ho buuoa ka boleng ba Tm ba primer, ho kgothaletswa ho beha mocheso o kenang ho boleng bo bonyenyane ba Tm ba primer +3 ~ 5 ℃.

Mathata a Tloaelehileng le Litharollo

1.Ha ho likhoele tse reriloeng

1) Sehlahisoa sa lysis se feteletseng.Khetha palo e nepahetseng ka ho fetisisa ea template, hangata eseng ho feta 1/10 ea sistimi;

2) Saese e kholo haholo.Hlakola lysate ka makhetlo a 10 ebe o holisa, kapa fokotsa boholo ba sampole le lysis hape;

3) Mehlala ea lithane ha e ncha.Ho khothalletsoa ho sebelisa lisampole tse ncha tsa lisele;

4) Boleng ba pele bo bobe.Sebelisa genomic DNA ho holisa ho netefatsa boleng ba primer le ho ntlafatsa moralo oa primer.

2.Matlafatso a sa khetheheng

1) Thempereichara ea annealing e tlase haholo mme palo ea potoloho e phahame haholo.Eketsa mocheso oa annealing le ho fokotsa palo ea lipotoloho;

2) Khatiso ea template e phahame haholo.Fokotsa palo ea thempleite kapa hlapolla thempleite makhetlo a 10 ka mor'a amplification;

3) Litlhaloso tse mpe tsa primer.Ntlafatsa moralo oa primer.

Lintlha

1.E le ho qoba tšilafalo pakeng tsa lisampole, lisebelisoa tse ngata tsa lisampole li lokela ho lokisoa, 'me bokaholimo ba lisebelisoa bo ka hloekisoa ka 2% sodium hypochlorite solution kapa nucleic acid cleaner ka mor'a sampling e' ngoe le e 'ngoe haeba ho sebelisoa khafetsa ho hlokahala.

2.Ho khothaletsoa ho sebelisa li-tissue tse ncha tsa toeba, 'me molumo oa sampole ha oa lokela ho ba kholo haholo ho qoba ho ama liphetho tsa ho holisa.

3.Lysis Buffer e lokela ho qoba ho qhibiliha khafetsa, 'me e ka bolokoa ho 2 ~ 8℃ bakeng sa tšebeliso ea nako e khuts'oane e mengata.Haeba e bolokiloe mochesong o tlase, pula e ka hlaha, 'me e tlameha ho qhibiliha ka botlalo pele e sebelisoa.

4.PCR Mix e lokela ho qoba ho qhibiliha khafetsa, 'me e ka bolokoa ho 4℃ bakeng sa tšebeliso ea nako e khutšoane khafetsa.

5.Sehlahisoa sena ke sa lipatlisiso tsa mahlale feela 'me ha sea lokela ho sebelisoa ho hlahlojoa kapa ho phekoloa.