Mouse Genotyping Kit

Nambala ya mphaka: HCR2021A

Izi ndi zida zomwe zidapangidwira kuti zizindikire mwachangu ma genotypes a mbewa, kuphatikiza DNA crude extraction ndi PCR amplification system.Mankhwalawa atha kugwiritsidwa ntchito pakukulitsa kwa PCR molunjika kuchokera ku mchira wa mbewa, khutu, chala chala ndi minyewa ina pambuyo poduka mosavuta ndi Lysis Buffer ndi Proteinase k.Palibe chimbudzi chausiku, phenol-chloroform m'zigawo kapena kuyeretsa mzati, zomwe zimakhala zosavuta komanso zimafupikitsa nthawi yoyeserera.Chogulitsacho ndi choyenera kukulitsa zidutswa zomwe mukufuna mpaka 2kb ndi ma multiplex PCR ndi mapeyala atatu oyambira.2 × Mouse Tissue Direct PCR Mix ili ndi DNA polymerase, Mg.²⁺, ma dNTP ndi makina okhathamiritsa a buffer kuti apereke mphamvu zokulitsa bwino komanso kulolerana kwa inhibitor, kotero kuti machitidwe a PCR atha kuchitidwa powonjezera template ndi zoyambira ndikubwezeretsanso mankhwala ku 1 ×.Pulogalamu ya PCR yokulitsidwa ndi mankhwalawa ili ndi maziko odziwika bwino a "A" kumapeto kwa 3' ndipo angagwiritsidwe ntchito mwachindunji ku TA cloning pambuyo pa kuyeretsedwa.

Zigawo

Zosungirako

Zogulitsa ziyenera kusungidwa pa -25 ~ -15 ℃ kwa zaka 2.Pambuyo thawing, Lysis Buffer akhoza kusungidwa pa 2 ~ 8 ℃ yochepa ntchito angapo, ndi kusakaniza bwino ntchito.

Kugwiritsa ntchito

Izi ndizoyenera kuwunikira kugogoda kwa mbewa, kuzindikira kwa transgenic, genotyping ndi zina zotero.

Mawonekedwe

1.Ntchito yosavuta: palibe chifukwa chochotsera DNA ya genomic;

2.Lonse ntchito: oyenera kukulitsa mwachindunji zosiyanasiyana mbewa zimakhala.

Malangizo

1.Kutulutsidwa kwa DNA ya genomic

1) Kukonzekera kwa lysate

Minofu ya lysate imakonzedwa molingana ndi kuchuluka kwa zitsanzo za mbewa zomwe zimayenera kupangidwa (minofu ya lysate iyenera kukonzedwa pamalopo molingana ndi mlingo ndikusakaniza bwino kuti mugwiritse ntchito), ndipo kuchuluka kwa ma reagents ofunikira pa chitsanzo chimodzi ndi motere:

2) Kukonzekera Zitsanzo ndi Lysis

Kugwiritsiridwa ntchito kwa Tissue

Tengani kuchuluka koyenera kwa zitsanzo za minofu ya mbewa mu machubu oyera a centrifuge, onjezerani 200μL wa minofu yatsopano lysate ku chubu chilichonse cha centrifuge, vortex ndi kugwedeza, ndiye muyimire pa 55 ℃ kwa 30mins, ndiyeno kutentha pa 98 ℃ kwa 3mins.

3) Centrifugation

Gwirani bwino lysate ndi centrifuge pa 12,000 rpm kwa 5mins.Zowonjezereka zitha kugwiritsidwa ntchito ngati template yakukulitsa PCR.Ngati template ikufunika kuti isungidwe, tumizani chowonjezeracho ku chubu china chosabala cha centrifuge ndikusunga pa -20 ℃ kwa milungu iwiri.

2.Kusintha kwa PCR

Chotsani 2 × Mouse Tissue Direct PCR Mix kuchokera ku -20 ℃ ndikusungunula pa ayezi, sakanizani mozondoka ndikukonzekera dongosolo la PCR molingana ndi tebulo ili (ntchito pa ayezi):

Zindikirani:

a) Ndalama zomwe zawonjezeredwa siziyenera kupitirira 1/10 ya dongosolo, ndipo ngati zambiri zawonjezeredwa, kukulitsa kwa PCR kungakhale koletsedwa.

Zovomerezeka za PCR

Zindikirani:

a) Kutentha kwa Annealing: Ponena za mtengo wa Tm wa primer, tikulimbikitsidwa kukhazikitsa kutentha kwa annealing ku mtengo wocheperako wa Tm wa primer +3 ~ 5 ℃.

Mavuto Wamba ndi Mayankho

1.Palibe mizere yolunjika

1) Kuchuluka kwa lysis mankhwala.Sankhani kuchuluka koyenera kwa template, nthawi zambiri osapitirira 1/10 ya dongosolo;

2) Kukula kwakukulu kwachitsanzo.Kuchepetsa lysate nthawi 10 ndiyeno kukulitsa, kapena kuchepetsa chitsanzo kukula ndi kukonzanso lysis;

3) Zitsanzo za minofu si zatsopano.Ndibwino kugwiritsa ntchito zitsanzo za minofu yatsopano;

4) Zoyambira zabwino kwambiri.Gwiritsani ntchito genomic DNA pakukulitsa kuti mutsimikizire mtundu wa choyambirira ndikuwongolera kapangidwe kake.

2.Kukulitsa kosakhazikika

1) Kutentha kwa annealing ndikotsika kwambiri ndipo nambala yozungulira ndiyokwera kwambiri.Wonjezerani kutentha kwa annealing ndikuchepetsa kuchuluka kwa kuzungulira;

2) Kuyika kwa template ndikokwera kwambiri.Chepetsani kuchuluka kwa template kapena kuchepetsa template nthawi 10 mutatha kukulitsa;

3) Kusakhazikika koyambirira.Konzani mapangidwe oyambira.

Zolemba

1.Pofuna kupewa kuipitsidwa pakati pa zitsanzo, zida zingapo zoyeserera ziyenera kukonzedwa, ndipo pamwamba pazidazo zitha kutsukidwa ndi 2% sodium hypochlorite solution kapena nucleic acid cleaner pambuyo pa sampuli iliyonse ngati ikufunika kugwiritsidwa ntchito mobwerezabwereza.

2.Ndikoyenera kugwiritsa ntchito mbewa zatsopano, ndipo voliyumu yachitsanzo isakhale yayikulu kwambiri kuti isakhudze zotsatira zakukulitsa.

3.Lysis Buffer iyenera kupewa kuzizira pafupipafupi, ndipo imatha kusungidwa pa 2 ~ 8 ℃ kuti igwiritsidwe ntchito kwakanthawi kochepa.Ngati kusungidwa pa kutentha kochepa, mvula imatha kuchitika, ndipo iyenera kusungunuka musanagwiritse ntchito.

4.PCR Mix iyenera kupewa kuzizira pafupipafupi, ndipo imatha kusungidwa pa 4 ℃ kuti igwiritsidwe ntchito mobwerezabwereza.

5.Izi ndi zongoyesa kafukufuku wasayansi ndipo siziyenera kugwiritsidwa ntchito pozindikira matenda kapena kuchiza.