TIANSeq HiFi Amplification Kusakaniza

Kukulitsa laibulale PCR premix yokhala ndi zokolola zambiri mulaibulale, kukhulupirika kwambiri komanso kukondera pang'ono.

TIANSeq HiFi Ampli ation Mix ndichinthu chatsopano chotsimikizika cha kukhulupirika kwa PCR premix, chomwe chili choyenera kukweza kwa PCR kwa laibulale yotsatizana bwino kwambiri komanso zina zofananira ndi PCR. Ultra HiFi DNA Polymerase mu yankho lisanaphatikizidwe ndi mtundu watsopano wachangu komanso wokhulupirika kwambiri wa DNA polymerase wopangidwa ndi ukadaulo wamakanema wopanga, womwe umalimbikitsa kuyanjana kwa DNA polymerase ndi ma tempuleti. Enzyme iyi yakulitsa kuthamanga kwamphamvu komanso kukulitsa kuthekera, ndipo kuchuluka kwa PCR ndi zokolola zamagetsi kukuwonjezeka. Enzyme imakhalanso ndi zochitika zabwino kwambiri za 3'- 5 ′ (Kuchita zowerengera), ndipo kudalirika kwake kumatha kufikira pamsika wazogulitsa, kutsimikizika kwake pakupanga majini ndikukweza laibulale. Kuphatikiza apo, DNA polymerase yomwe imagulitsidwayo ili ndi HotStart function, yomwe imatha kuwongolera kutulutsa kosakhazikika komanso kutayika kwa ma enzyme pansi pa kutentha kocheperako, motero kuwonetsetsa kuti PCR ikukulira komanso kukhazikika. Makina opangira ma PCR amaphatikizidwanso mu Kusakanikirana, komwe sikungowonjezera kusakanikirana kwa Kusakanikirana, kumathandizira kulolerana kwa ma polymerase ku PCR reaction inhibitors, komanso kumathandizira kusintha kwa Ultra HiFi DNA Polymerase pamatchulidwe okhala ndi ma GC osiyanasiyana, kotero kuti malonda ali ndi zitsanzo zambiri zakuthambo komanso zoyeserera zoyeserera, ndikuchepetsa zomwe amakonda PCR.

Mphaka. Ayi	Kukulitsa Kukula
4992369	1 ml
4992370	5 × 1 ml
4992371	5 * 1ml
4992372	5 * 1ml

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FAQ

Zogulitsa

Kumunsi

Kukulitsa kwa laibulale ya NGS PCR, kukulitsa kwa m'badwo woyamba kutsata kwa PCR, kukhulupirika kwakukulu, kuzindikira kwa SNP, kusintha kwa tsamba, etc.

Mawonekedwe

■ Kukulitsa kwamphamvu kwambiri: Onetsetsani kusinthaku ndikuchepetsa magwiridwe antchito.
■ Kukonda Kwambiri: Kukhathamiritsa koyenera kwamatemplate a DNA okhala ndi GC% zosiyanasiyana.
■ Kutsogola kwapamwamba: Ndi malo a HotStart ndikudziwika kwamphamvu.
■ Kukhulupirika kwakukulu: Kukhulupirika kumakhala kowirikiza 50 kuposa Taq DNA polymerase.
■ Kuzindikira kwambiri: Kulowetsa ma template kumatha kutsika 1 pg.

Zogulitsa zonse zimatha kusinthidwa kukhala ODM / OEM. Zambiri,chonde dinani Makonda Service (ODM / OEM)

Previous: Zamgululi TIANSeq Adapter Yokha-Index (Illumina)

Ena: TIANSeq Chidutswa / Kukonza / Kuyika Module

	Chithunzi 1. Kupititsa patsogolo laibulale ya genomic DNA ndi magawanidwe osiyanasiyana a GC (genomic input 10 ng, 8 cycles amplification) idachitika nthawi imodzi pogwiritsa ntchito TIANSeq HiFi Amplification Mix ndi HiFi enzyme yochokera ku Supplier K ndi N, ndipo zokolola mu laibulale zidapezeka ndi Agilent Zotsatira za 2100. Zotsatira zake zidawonetsa kuti TIANSeq HiFi Amplification Mix ili ndi zokolola zochuluka mulaibulale, yokhala ndi mphamvu ponseponse pazosiyanasiyana za GC, ndipo magwiridwe antchito opangira laibulale anali bwino kuposa ena ogulitsa.
	Kufufuza detaGwiritsani ntchito TIANSeq HiFi Amplification Mix ndi ma enzyme a HiFi omwe amagwiritsidwa ntchito makamaka kukulitsa laibulale ya NGS kuchokera kwa Supplier K ndi N pokweza laibulale ya genomic yomweyo ya DNA (ma genome input ndi 10 ng). Mukamaliza kusanja, pendani kuchuluka ndi kubwereza kwa laibulale.
Kukonda kwa GC	Chithunzi 2. Kwezani malaibulale a genome okhala ndi zinthu zosiyanasiyana za CG pogwiritsa ntchito TIANSeq HiFi Amplification Mix ndi HIFi yochokera ku Supplier K ndi N. Zotsatira zake zikuwonetsa kuti kufanana kwa laibulale ya TIANSeq HiFi Amplification Mix ndi yabwino komanso yopanda GC, zomwe ndizofanana ndi zotsatira ya kampani K komanso yabwinoko pang'ono kuposa zomwe kampani yakampani N. Zotsatirazo zikuwonetsa kuti kufotokozedwa kwa laibulale ya TIANSeq HiFi Amplification Mix amplification ndiyokwera, kuchuluka kwakubwereza kumakwaniritsa zofunikira, ndipo magwiridwe antchito a laibulale amafanana ndi omwe akupikisana nawo.

Q: Kodi kugawa kwa zidutswa zonse mu library ya NGS ndikuti?

Pakadali pano, ukadaulo wopanga wapamwamba kwambiri umakhazikitsidwa makamaka paukadaulo wazotsatira za m'badwo wotsatira. Popeza kutalika kwa kuwerenga kwaukadaulo wazotsatira zam'badwo wotsatira kuli kochepa, tiyenera kudula magawo athunthu kukhala malaibulale ang'onoang'ono motsatizana. Malinga ndi zosowa zoyeserera mosiyanasiyana, nthawi zambiri timasankha kusanja komwe kumatha kamodzi kapena kutsata kawiri. Pakadali pano zidutswa za DNA zam'buku lotsatira lotsatira laibulale zimagawidwa mu 200-800 bp.

Q: Kuchuluka kwa DNA mulaibulale yomwe yamangidwa ndikotsika.

a) DNA ndi yopanda phindu ndipo imakhala ndi zoletsa. Gwiritsani ntchito zitsanzo za DNA zapamwamba kwambiri kuti mupewe kuletsa michere.

b) Kuchuluka kwa zitsanzo za DNA sikukwanira mukamagwiritsa ntchito njira yopanda PCR popanga laibulale ya DNA. Pomwe kulowetsedwa kwa DNA yogawanika kukupitilira 50 ng, mayendedwe opanda ntchito a PCR amatha kuchitidwa panthawi yomanga laibulale. Ngati kuchuluka kwa laibulale kuli kotsika kwambiri kuti singathetsedwe mwachindunji, laibulale ya DNA imatha kukulitsidwa ndi PCR pambuyo pa adapter ligation.

c) Kuwonongeka kwa RNA kumabweretsa kuwonongeka koyambirira kwa DNA kukhathamiritsa kwa RNA kumatha kupezeka pakuyeretsa kwa genomic DNA, komwe kumatha kubweretsa kulakwitsa kwa DNA ndikusakwanira kwa DNA pakumanga laibulale. RNA ikhoza kuchotsedwa pochiza ndi RNase.

Q: Laibulale ya DNA idawonetsa magulu osadziwika pakuwunika kwa electrophoresis.

A-1

a) Zidutswa tating'ono (60 bp-120 bp) zimawoneka Zidutswa zing'onozing'ono nthawi zambiri zimakhala zidutswa zama adapta kapena ma dimers opangidwa ndi ma adapter. Kuyeretsa ndi Agencourt AMPure XP mikanda yamaginito kumatha kuchotsa zidutswazo ndikuwonetsetsa kuti ndiyabwino.

b) Zidutswa zazikulu zimapezeka mulaibulale pambuyo pa kukweza kwa PCR Kukula kwa chidutswa cha laibulale ya DNA kudzawonjezeka ndi 120 bp adaputala atalumikiza. Ngati chidutswa cha DNA chikuchulukirachulukira kuposa 120 bp pambuyo pa adapter ligation, itha kuyambika chifukwa chakuwonjezera pang'ono kwa PCR kukulitsa. Kuchepetsa kuchuluka kwa ma PCR kumatha kuletsa izi.

c) Kukula kwakukulu kwa zidutswa za library za DNA pambuyo pa adapter ligation Kutalika kwa adapter mu kit iyi ndi 60 bp. Mbali zonse ziwiri za chidutswacho zikalumikizidwa ndi ma adapter, kutalika kumangowonjezeka ndi 120 bp. Mukamagwiritsa ntchito adaputala kupatula yomwe idaperekedwa ndi chida ichi, chonde lemberani kwa woperekayo kuti akupatseni zambiri monga kutalika kwa adapter. Chonde onetsetsani kuti mayendedwe oyeserera ndi ntchito zikutsatira njira zomwe zafotokozedwamo.

d) Kukula kwazigawo zazing'ono za DNA pamaso pa adapter ligation Chomwe chimayambitsa vutoli chingachitike chifukwa chazomwe zimachitika panthawi yopatukana kwa DNA. Nthawi zosiyanasiyana zimayenera kugwiritsidwa ntchito popanga ma DNA osiyanasiyana. Ngati kulowetsa kwa DNA kuli kopitilira 10 ng, tikukulimbikitsani kuti musankhe nthawi yochitira mphindi 12 ngati nthawi yoyambira kukhathamiritsa, ndipo kukula kwa zidutswa zomwe zimapangidwa panthawiyi makamaka kuli 300-500 bp. Ogwiritsa ntchito amatha kukulitsa kapena kuchepetsa kutalika kwa zidutswa za DNA kwa mphindi 2-4 malinga ndi zofuna zawo kuti akwaniritse zidutswa za DNA ndi kukula kofunikira.

A-2

a) Nthawi yogawanika siyabwino Ngati DNA yogawanika ndi yaying'ono kwambiri kapena yayikulu kwambiri, chonde onani Maupangiri a Kusankhidwa kwa Nthawi Yogawika omwe aperekedwa kuti athe kudziwa nthawi yoyankha, ndikugwiritsanso ntchito nthawi iyi ngati chiwongolero, kuwonjezera kukhazikitsa njira yothetsera kutalika kapena kufupikitsa 3 min kuti musinthe moyenera nthawi yogawanika.

A-3

Kukula kwakukulu kwa DNA patatha mankhwala ogawanika

a) Njira yolakwika yogawanitsira reagent, kapena reagent siyosakanikirana itasungunuka. Thaw 5 × Fragmentation Enzyme Mix reagent pa ayezi. Mukasungunuka, sakanizani reagent mofanana mwa kutsegula pang'onopang'ono pansi pa chubu. Osatengera vortex reagent!

b) Zoyeserera za DNA zili ndi EDTA kapena zoipitsa zina Kutha kwa ayoni amchere ndi oonera mu gawo la kuyeretsa kwa DNA ndikofunikira kwambiri kuti kuyesaku kuyende bwino. Ngati DNA isungunuka mu 1 × TE, gwiritsani ntchito njira yomwe yaperekedwayo popanga kugawanika. Ngati kusakanikirana kwa EDTA mu yankho sikudziwika, tikulimbikitsidwa kuyeretsa DNA ndikuisungunula m'madzi osakanikirana kuti muthe kuyankha.

c) Kulondola kolondola koyamba kwa DNA Kukula kwa DNA yogawanika kumagwirizana kwambiri ndi kuchuluka kwa kulowa kwa DNA. Asanalandire chithandizo, kugawanika molondola kwa DNA pogwiritsa ntchito Qubit, Picogreen ndi njira zina ndikofunikira kudziwa kuchuluka kwa DNA mumachitidwe.

d) Kukonzekera kwa zomwe zimachitika sikutsatira malangizowo Kukonzekera kwa magawano oyanjana kuyenera kuchitidwa pa ayezi mosamalitsa malinga ndi malangizo. Kuti muwonetsetse kuti zinthu zikuyenda bwino, zonse zomwe zimayankhidwa ziyenera kuikidwa pa ayezi ndikukonzekera njira yochitira pambuyo pozizira kwathunthu. Mukamaliza kukonzekera, chonde flick kapena pipet kuti musakanize bwino. Osati vortex!

Q: Zolemba zofunikira pa TIANSeq DirectFast DNA Library Kit (Illumina) (4992259/4992260)

1. Njira zosakanikirana bwino (vortex, oscillation yachiwawa, ndi zina zambiri) zitha kuyambitsa kugawidwa kwachilendo kwa zidutswa za laibulale (monga zikuwonetsedwa pachithunzipa), zomwe zimakhudza laibulale. Chifukwa chake, pokonzekera mayankho a Fragmentation Mix, chonde mokweza mmwamba ndi pansi kuti musakanize, kapena gwiritsani ntchito chala chakumanja kuti muzungulire ndikusakanikirana mofanana. Samalani kuti musasakanikirane ndi vortex.

2. DNA yoyera kwambiri iyenera kugwiritsidwa ntchito popanga laibulale

■ Umphumphu wabwino wa DNA: Gulu la electrophoresis limaposa 30 kb, popanda kuchira

■ OD260 / 230:> 1.5

■ OD260 / 280: 1.7-1.9

3. Kuchulukitsa kwa DNA kuyenera kukhala kolondola Zimanenedwa kuti mugwiritse ntchito njira za Qubit ndi PicoGreen kuti muyese DNA, osati Nanodrop.

4. Zomwe zili mu EDTA mu yankho la DNA ziyenera kutsimikiziridwa kuti EDTA imakhudza kwambiri magawanidwewa. Ngati zomwe zili mu EDTA ndizokwera, kuyeretsa kwa DNA kuyenera kuchitidwa mayeso asanakwane.

5. Njira yothetsera mavutowo iyenera kukonzedwa pa ayezi Njira yogawanikana ndiyofunika kuzindikira kutentha ndi nthawi (makamaka mutatha kuwonjezera zowonjezera). Pofuna kuonetsetsa kuti nthawi ikuyenda molondola, chonde konzekerani momwe mungayankhire pa ayezi.

6. Nthawi yogawika iyenera kukhala yolondola Nthawi yomwe gawo logawanika lingakhudze kukula kwa zopangidwa, zomwe zingakhudze kukula kwa magawo a DNA mulaibulale.

Q: Zolemba zofunikira za TIANSeq Fast RNA Library Kit (Illumina) (4992375/4992376)

1. Kodi ndi zitsanzo ziti zomwe zikugwiritsidwa ntchito pa chida ichi?

Mtundu woyenera wa chida ichi ukhoza kukhala RNA yathunthu kapena mRNA yoyeretsedwa yokhala ndi umphumphu wabwino wa RNA. Ngati RNA yonse imagwiritsidwa ntchito popanga laibulale, tikulimbikitsidwa kuti mugwiritse ntchito rRNA depletion kit (Cat # 4992363/4992364/4992391) kuti muchotse rRNA kaye.

2. Kodi FFPE ingagwiritsidwe ntchito popanga laibulale ndi chida ichi?

MRNA mu zitsanzo za FFPE zidzawonongeka pamlingo winawake, ndi umphumphu wochepa. Mukamagwiritsa ntchito chida ichi popanga laibulale, tikulimbikitsidwa kuti tithandizire nthawi yogawika (kufupikitsa nthawi yogawikana kapena osagawika).

3. Pogwiritsa ntchito sitepe yosankha kukula koperekedwa m'buku lazogulitsa, ndi chiyani chomwe chingapangitse kuti gawo lolowetsedwa liwoneke ngati likupatuka pang'ono?

Kusankha kwamiyeso kumayendetsedwa molingana ndi sitepe yosankha kukula kwa bukuli. Ngati pali kupatuka, chifukwa chake mwina maginito amikono siabwino kutentha kwa firiji kapena sanasakanikirane bwino, pipette siyolondola kapena madzi amakhalabe kumapeto. Ndikulimbikitsidwa kuti mugwiritse ntchito malangizowo okhala ndi zotsatsa zochepa poyesa.

4. Kusankha ma adapter mu zomangamanga

Zida zomangira laibulale sizikhala ndi reagent ya adapta, ndipo tikulimbikitsidwa kuti tigwiritse ntchito chida ichi pamodzi ndi TIANSeq Single-Index Adapter (Illumina) (4992641/4992642/4992378).

5. QC ya laibulale

Kuzindikira kuchuluka kwa Library: Qubit ndi qPCR amagwiritsidwa ntchito kuti azindikire kuchuluka kwa kuchuluka kwa laibulale motsatira. Ntchitoyi ikugwirizana ndi buku lazogulitsa. Kuchuluka kwa laibulale kudzakwaniritsa zofunikira za kutsata kwa NGS. Kudziwika kwa magawidwe amulaibulale: Kugwiritsa ntchito Agilent 2100 Bioanalyzer kuti mupeze magawo ogawa laibulale.

6. Kusankhidwa kwa kuchuluka kwamazenera

Malinga ndi malangizowo, kuchuluka kwa ma PCR ndi 6-12, ndipo kuchuluka kwa ma PCR oyenera kuyenera kusankhidwa malinga ndi mayendedwe achitsanzo. M'malaibulale okhala ndi zokolola zambiri, kupititsa patsogolo kwamphamvu nthawi zambiri kumachitika mosiyanasiyana, komwe kumawonetsedwa ndi nsonga yayikulu pang'ono pambuyo pachimake cha chandamale pozindikira Agilent 2100 Bioanalyzer, kapena kuchuluka kwa Qubit ndikotsika kuposa kwa qPCR. Kufatsa kwamphamvu ndikumveka bwino, komwe sikungakhudze kutsata kwa laibulale ndikusanthula kwotsatira kwa deta.

7. Ma spikes amawoneka mu mbiri yodziwika ya Agilent 2100 Bioanalyzer

Maonekedwe a ma spikes pakuzindikira kwa Agilent 2100 Bioanalyzer ndichifukwa chakugawika kosagwirizana kwa zitsanzo, pomwe padzakhala tizidutswa tambiri kukula kwake, ndipo izi zimawonekeranso pambuyo pakukula kwa PCR. Poterepa, akuti sakusankha kukula, mwachitsanzo, khazikitsani magawikidwe kukhala 94 ° C kwa mphindi 15 yoyambitsidwa, pomwe magawidwe ake amakhala ochepa komanso okhazikika, ndipo kusakanikirana kumatha kukonzedwa.

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