TIANSeq HiFi Amplification Mix

Lub tsev qiv ntawv nthuav dav PCR ua ntej nrog cov tsev qiv ntawv tau txais txiaj ntsig zoo, muaj kev ntseeg siab thiab muaj lub hauv paus qis.

TIANSeq HiFi Ampli ﬁ cation Mix yog qhov tshiab fidelity PCR kev nthuav dav ua ntej, uas yog qhov tsim nyog rau PCR kev nthuav dav ntawm kev nkag mus tau zoo hauv lub tsev qiv ntawv thiab lwm yam PCR cuam tshuam nrog cloning thiab nrhiav pom. Ultra HiFi DNA Polymerase nyob rau hauv kev daws ua ntej sib xyaw yog hom tshiab ntawm kev nrawm thiab siab ncaj DNA polymerase tsim los ntawm kev qhia thev naus laus zis hloov pauv thev naus laus zis, uas pab txhawb kev sib raug zoo ntawm DNA polymerase rau cov qauv. Cov enzyme no tau txhim kho kev nthuav dav nrawm thiab muaj peev xwm txuas ntxiv, thiab kev ua tiav ntawm PCR thiab cov khoom lag luam tau nce ntxiv. Cov enzyme kuj tseem muaj qhov ua tau zoo 3′- 5 ′ exonuclease (Kev txheeb xyuas qhov ua haujlwm), thiab nws qhov kev ncaj ncees tuaj yeem ncav cuag qib ntawm cov khoom lag luam tseem ceeb hauv khw, nrog qhov tseeb ntawm cov txheej txheem ntawm cov noob thiab tsev qiv ntawv nthuav dav kom ntseeg tau. Ib qho ntxiv, DNA polymerase hauv cov khoom muaj qhov ua haujlwm HotStart, uas tuaj yeem tswj tau qhov tsis tshwj xeeb nthuav dav thiab kev ua haujlwm enzyme poob qis hauv qhov kub, yog li ua kom muaj kev tshwj xeeb thiab ruaj khov ntawm PCR kev nthuav dav. PCR cov tshuaj tiv thaiv kev txhim kho kuj tseem muaj kev sib xyaw, uas tsis tsuas yog txhim kho kev ruaj ntseg ntawm Kev Sib xyaw, txhim kho kev ua siab ntev ntawm polymerase rau PCR cov tshuaj tiv thaiv kab mob, tab sis tseem txhim kho kev hloov pauv ntawm Ultra HiFi DNA Polymerase rau cov qauv nrog GC cov ntsiab lus sib txawv, yog li tias cov khoom lag luam muaj cov qauv dav dav thoob ntiaj teb thiab cov qauv ua piv txwv ntau, thiab txo qis qhov kev nyiam ntawm PCR cov tshuaj tiv thaiv.

Miv. Tsis yog	Ntim Loj
4992369	1ml ua
4992370	5 × 1 ml
4992371	5*1 hli
4992372	5*1 hli

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NGS lub tsev qiv ntawv PCR kev nthuav dav, 1st-generation sequencing PCR amplification, siab ncaj ncees cloning, SNP kuaj pom, hloov chaw tshwj xeeb, thiab lwm yam.

Nta

-Kev ua kom muaj txiaj ntsig zoo: Ua kom muaj kev hloov pauv thiab txo qis kev ua haujlwm.
■ Kev Nyuaj Siab Nyiam: Sib npaug ua kom muaj txiaj ntsig zoo rau DNA cov qauv nrog GC% cov ntsiab lus sib txawv.
Specific Tshwj xeeb tshwj xeeb: Nrog HotStart cov cuab yeej thiab muaj zog tshwj xeeb.
■ Siab ncaj ncees: Kev ncaj ncees yog 50 npaug siab dua Taq DNA polymerase.
■ Kev nkag siab siab: Cov tswv yim nkag tuaj yeem qis li 1 pg.

Txhua yam khoom tuaj yeem tsim kho rau ODM/OEM. Yog xav paub ntxiv,thov nyem Customized Service (ODM/OEM)

Yav dhau los: TIANSeq Ib-Index Adapter (Illumina)

Tom ntej: TIANSeq Tshooj/Kho/Tailing Module

	Daim duab 1. Lub tsev qiv ntawv txhim kho cov genomic DNA nrog GC sib piv sib txawv (cov tswv yim genomic 10 ng, 8 cycles amplification) tau ua tiav ib txhij los ntawm kev siv TIANSeq HiFi Amplification Mix thiab HiFi enzyme los ntawm Tus K thiab N, thiab cov tsev qiv ntawv tau pom los ntawm Agilent 2100. Cov txiaj ntsig tau qhia tias TIANSeq HiFi Amplification Mix muaj cov tsev qiv ntawv tau zoo, nrog rau kev muaj zog thoob ntiaj teb rau cov ntsiab lus sib txawv ntawm GC, thiab kev ua kom lub tsev qiv ntawv ua tau zoo dua li lwm tus neeg muab khoom.
	Sequencing cov ntaub ntawvSiv TIANSeq HiFi Amplification Mix thiab HiFi enzyme tshwj xeeb siv rau NGS lub tsev qiv ntawv nthuav dav los ntawm Tus Muab K thiab N rau lub tsev qiv ntawv nthuav dav ntawm tib lub genomic DNA (genome input yog 10 ng). Tom qab ua ntu zus, txheeb xyuas qhov kev pab them nqi thiab qhov sib npaug ntawm cov tsev qiv ntawv.
Kev nyiam GC	Daim duab 2. Ua kom cov tsev qiv ntawv genome sib txawv nrog CG cov ntsiab lus siv TIANSeq HiFi Amplification Mix thiab HIFi los ntawm Cov Khoom K thiab N. ntawm lub tuam txhab K thiab me ntsis zoo dua li cov khoom lag luam ntawm tuam txhab N. Cov txiaj ntsig tau qhia tias kev pab them nqi ntawm TIANSeq HiFi Amplification Mix amplification library yog siab, qhov kev theej tawm tau ua tau raws li qhov xav tau, thiab kev ua haujlwm ntawm lub tsev qiv ntawv yog sib npaug ntawm cov neeg sib tw.

Q: Dab tsi yog qhov kev faib dav dav ntawm qhov faib me me hauv NGS lub tsev qiv ntawv?

Tam sim no, kev siv tshuab thev naus laus zis siab yog feem ntau yog ua raws cov thev naus laus zis tom ntej. Raws li kev nyeem ntawv ntev ntawm cov thev naus laus zis sib txuas tom ntej muaj qhov txwv, peb yuav tsum ua txhaum tag nrho cov ntu ua ntu ntev mus rau hauv cov tsev qiv ntawv me me ua ntu zus. Raws li qhov xav tau ntawm kev sim ua ntu sib txawv, peb feem ntau xaiv ib leeg-ua ntu lossis ob-ntu ua ntu zus. Tam sim no cov DNA tawg ntawm cov tsev qiv ntawv sib txuas tom ntej no feem ntau raug faib hauv thaj tsam ntawm 200-800 bp.

Q: DNA concentration ntawm lub tsev qiv ntawv tsim tsev tsawg.

a) DNA tsis zoo thiab muaj cov tshuaj tiv thaiv kab mob. Siv cov qauv DNA zoo los tiv thaiv kev ua haujlwm ntawm enzyme.

b) Tus nqi ntawm cov qauv DNA tsis txaus thaum siv PCR-tsis muaj txoj hauv kev los tsim cov tsev qiv ntawv DNA. Thaum cov tswv yim ntawm cov DNA tawg ua ntau dua 50 ng, PCR-tsis ua haujlwm tuaj yeem xaiv tau ua thaum lub sijhawm tsim cov tsev qiv ntawv. Yog tias daim ntawv theej ntawm lub tsev qiv ntawv qis dhau los ua ntu ntu ncaj qha, lub tsev qiv ntawv DNA tuaj yeem nthuav dav los ntawm PCR tom qab hloov kho qhov txuas.

c) RNA kev sib kis ua rau tsis raug tus lej DNA thawj zaug RNA kev sib kis tuaj yeem muaj nyob hauv cov txheej txheem kev ua kom huv ntawm cov genomic DNA, uas tuaj yeem ua rau DNA suav tsis raug thiab tsis txaus DNA thauj khoom thaum tsim tsev qiv ntawv. RNA tuaj yeem tshem tawm los ntawm kev kho nrog RNase.

Q: Lub tsev qiv ntawv DNA pom cov pab pawg tsis txawv txav hauv kev tshuaj xyuas hluav taws xob.

A-1

a) Cov khoom me me (60 bp-120 bp) zoo li cov khoom me me feem ntau yog cov khoom tawg lossis cov dimers tsim los ntawm cov hloov pauv. Kev ua kom huv nrog Agencourt AMPure XP sib nqus hlaws dai tuaj yeem tshem tawm cov khoom tawg no kom zoo thiab ua kom muaj kev sib dhos zoo.

b) Cov tawg loj tshwm nyob rau hauv lub tsev qiv ntawv tom qab PCR kev nthuav dav Qhov loj ntawm lub tsev qiv ntawv DNA tawg ua ke yuav nce ntxiv 120 bp tom qab lub adapter txuas nrog. Yog tias DNA qhov tawg tau nce ntau dua 120 bp tom qab hloov kho qhov txuas, nws yuav tshwm sim los ntawm qhov txawv txav ntawm kev faib tawm ntawm PCR ntau dhau. Txo cov naj npawb ntawm PCR mus los tuaj yeem tiv thaiv qhov xwm txheej.

c) Qhov txawv me me ntawm cov tsev qiv ntawv DNA tawg tom qab hloov kho qhov ntev Qhov hloov pauv hauv cov khoom siv no yog 60 bp. Thaum ob qhov kawg ntawm qhov tawg tau sib txuas rau cov hloov pauv, qhov ntev yuav tsuas yog nce ntxiv 120 bp. Thaum siv lub adapter tsis yog qhov uas tau muab los ntawm cov khoom siv no, thov hu rau tus muag khoom kom muab cov ntaub ntawv cuam tshuam xws li qhov ntev adapter. Thov kom ntseeg tau tias kev sim ua haujlwm thiab ua haujlwm ua raws cov kauj ruam tau piav qhia hauv phau ntawv qhia.

d) Qhov txawv me me ntawm DNA qhov loj me ua ntej hloov pauv adapter Vim li cas rau qhov teeb meem no tuaj yeem tshwm sim los ntawm cov xwm txheej tsis raug thaum lub sijhawm DNA tawg. Lub sijhawm sib txawv ntawm cov tshuaj tiv thaiv yuav tsum tau siv rau kev nkag DNA sib txawv. Yog tias DNA nkag mus ntau dua 10 ng, peb pom zoo kom xaiv lub sijhawm tshuaj tiv thaiv ntawm 12 min raws li lub sijhawm pib rau kev ua kom zoo dua, thiab qhov tawg me me uas ua rau lub sijhawm no feem ntau yog nyob ntawm 300-500 bp. Cov neeg siv tuaj yeem nce lossis txo qhov ntev ntawm DNA tawg rau 2-4 feeb raws li lawv tus kheej xav tau txhawm rau ua kom zoo dua cov DNA tawg nrog qhov xav tau loj.

A-2

a) Fragmentation time is not optimized If the fragmented DNA is too small or too big, please refer to the Guidelines for Fragmentation Time Selection provided in the instruction to determine the reaction time, and use this time point as a control, ntxiv rau teeb tsa cov txheej txheem tshuaj tiv thaiv kom ncua lossis luv 3 feeb kom hloov kho kom raug dua ntawm lub sijhawm tawg.

A-3

Qhov txawv me me faib ntawm DNA tom qab kev kho ua kom tawg

a) Txoj hauv kev yaj tsis raug ntawm kev faib cov tshuaj reagent, lossis cov tshuaj reagent tsis sib xyaw tom qab yaj tag. Thaw 5 × Fragmentation Enzyme Mix reagent ntawm dej khov. Thaum thawed, sib tov cov tshuaj reagent los ntawm maj mam ua rau hauv qab ntawm lub raj. Tsis txhob vortex lub reagent!

b) Cov qauv DNA suav nrog EDTA lossis lwm yam kuab paug Depletion ntsev ions thiab chelating cov neeg sawv cev hauv qib DNA kev ua kom huv yog qhov tseem ceeb tshwj xeeb rau kev sim ua tiav. Yog tias DNA tau yaj hauv 1 × TE, siv txoj hauv kev uas tau qhia hauv cov lus qhia kom ua kom tawg. Yog tias EDTA cov ntsiab lus hauv kev daws teeb meem tsis meej, nws raug nquahu kom ntxuav cov DNA thiab yaj nws hauv cov dej deionized rau cov tshuaj tiv thaiv tom ntej.

c) Thawj qhov tsis raug ntawm DNA qhov loj me ntawm DNA tawg ua feem sib cuam tshuam nrog cov nyiaj ntawm DNA nkag. Ua ntej kho qhov kev faib ua feem, ntsuas kom raug ntawm DNA siv Qubit, Picogreen thiab lwm txoj hauv kev yog qhov tseem ceeb los txiav txim siab pes tsawg ntawm DNA hauv cov tshuaj tiv thaiv kab mob.

d) Kev npaj ntawm cov tshuaj tiv thaiv kab mob tsis ua raws li kev qhia Kev npaj ua cov txheej txheem ua kom tawg yuav tsum tau ua ntawm cov dej khov nruj me ntsis raws li cov lus qhia. Yuav kom ntseeg tau tias muaj txiaj ntsig zoo tshaj plaws, txhua yam tshuaj tiv thaiv yuav tsum tau muab tso rau hauv dej khov thiab kev npaj cov tshuaj tiv thaiv kab mob yuav tsum tau ua tom qab ua kom txias. Tom qab kev npaj ua tiav, thov flick lossis pipet kom sib xyaw kom huv. Tsis txhob vortex!

Q: Cov ntawv tseem ceeb rau TIANSeq DirectFast DNA Library Kit (Illumina) (4992259/4992260)

1. Txoj kev sib xyaw tsis raug (vortex, kev ua phem qias neeg, thiab lwm yam) yuav ua rau muaj kev faib khoom tsis txaus ntseeg ntawm cov tsev qiv ntawv tawg (raws li qhia hauv daim duab hauv qab no), yog li cuam tshuam rau lub tsev qiv ntawv zoo. Yog li ntawd, thaum npaj Fragmentation Mix cov tshuaj tiv thaiv, thov maj mam muab cov raj tso rau saum thiab nqes los sib xyaw, lossis siv tus ntiv tes ntiv tes los nplawm thiab sib tov sib npaug. Ceev faj tsis txhob sib xyaw nrog vortex.

2. High purity DNA yuav tsum tau siv rau kev tsim tsev qiv ntawv

■ Zoo DNA ncaj ncees: Cov electrophoresis band muaj ntau dua 30 kb, yam tsis muaj tus tis

D OD260/230:> 1.5

D OD260/280: 1.7-1.9

3. DNA cov nyiaj yuav tsum yog qhov tseeb Nws tau hais qhia kom siv Qubit thiab PicoGreen txoj hauv kev los ntsuas DNA, ntau dua li Nanodrop.

4. Cov ntsiab lus ntawm EDTA hauv DNA daws yuav tsum txiav txim siab EDTA muaj kev cuam tshuam loj rau cov tshuaj tiv thaiv tawg. Yog tias cov ntsiab lus ntawm EDTA siab dua, kev ua kom huv DNA yuav tsum tau ua ua ntej qhov kev xeem tom ntej.

5. Kev faib cov tshuaj tiv thaiv tawg yuav tsum tau npaj rau ntawm cov dej khov Cov txheej txheem ua kom tawg yog ua rau cov tshuaj tiv thaiv kub thiab sijhawm (tshwj xeeb yog tom qab ntxiv cov tshuaj ntxiv). Txhawm rau kom ntseeg tau qhov tseeb ntawm lub sijhawm tshuaj tiv thaiv, thov npaj cov tshuaj tiv thaiv kab mob ntawm cov dej khov.

6. Lub sij hawm cov tshuaj tiv thaiv tawg yuav tsum muaj tseeb Lub sij hawm cov tshuaj tiv thaiv ntawm kev faib ua pawg yuav cuam tshuam ncaj qha rau qhov loj ntawm cov khoom tawg, yog li cuam tshuam qhov faib loj ntawm DNA tawg hauv lub tsev qiv ntawv.

Q: Cov ntawv tseem ceeb rau TIANSeq Fast RNA Library Kit (Illumina) (4992375/4992376)

1. Hom kev piv txwv twg haum rau cov khoom siv no?

Cov qauv piv txwv ntawm cov khoom siv no tuaj yeem yog RNA tag nrho lossis ua kom huv mRNA nrog kev ntseeg RNA zoo. Yog tias tag nrho RNA tau siv los tsim lub tsev qiv ntawv, nws raug nquahu kom siv rRNA cov khoom ua kom ploj (Cat#4992363/4992364/4992391) kom tshem rRNA ua ntej.

2. Cov qauv FFPE puas tuaj yeem siv los tsim tsev qiv ntawv nrog cov khoom siv no?

MRNA hauv FFPE cov qauv yuav raug txo qis rau qee qhov, nrog rau kev txheeb ze tsis zoo. Thaum siv cov khoom siv no rau kev tsim tsev qiv ntawv, nws raug nquahu kom ua kom zoo dua lub sijhawm tawg (ua kom luv lub sijhawm tawg lossis tsis ua kom tawg).

3. Siv cov txheej txheem xaiv me me muab rau hauv phau ntawv qhia khoom, dab tsi tuaj yeem ua rau ntu ntu tso tawm kom pom qhov txawv me ntsis?

Kev xaiv qhov loj me yuav tsum tau ua raws nruj raws qib xaiv me me hauv phau ntawv qhia khoom no. Yog tias muaj qhov sib txawv, qhov laj thawj yuav yog vim cov hlaws sib nqus tsis sib npaug rau chav sov lossis tsis sib xyaw ua ke, cov raj xa dej tsis raug lossis cov kua tseem nyob hauv qhov taub. Nws raug nquahu kom siv cov lus qhia nrog kev nqus qis rau kev sim.

4. Xaiv adapters hauv kev tsim tsev qiv ntawv

Cov khoom siv tsim tsev qiv ntawv tsis muaj cov tshuaj reagent, thiab nws tau pom zoo kom siv cov khoom siv no ua ke nrog TIANSeq Ib Leeg-Index Adapter (Illumina) (4992641/4992642/4992378).

5. QC ntawm lub tsev qiv ntawv

Kev tshawb nrhiav ntau lub tsev qiv ntawv: Qubit thiab qPCR tau siv los txiav txim siab qhov hnyav thiab qhov mloog zoo ntawm lub tsev qiv ntawv feem. Kev ua haujlwm yog nruj me ntsis raws li phau ntawv qhia khoom. Kev tsom xam ntawm lub tsev qiv ntawv feem ntau yuav ua tau raws li qhov yuav tsum tau ua ntawm NGS. Tshawb nrhiav ntau lub tsev qiv ntawv faib tawm: Siv Agilent 2100 Bioanalyzer txhawm rau txheeb xyuas lub tsev qiv ntawv faib ntau yam.

6. Xaiv tus naj npawb ntawm kev hloov pauv

Raws li cov lus qhia, tus naj npawb ntawm PCR mus yog 6-12, thiab tus naj npawb ntawm PCR kev xav tau yuav tsum tau xaiv raws li cov qauv nkag. Hauv cov tsev qiv ntawv muaj txiaj ntsig zoo, dhau qhov ua kom nrov nrov feem ntau tshwm sim hauv qib sib txawv, uas tau tshwm sim los ntawm qhov nce siab me ntsis tom qab lub ncov ntawm lub hom phiaj ntau hauv kev tshawb pom ntawm Agilent 2100 Bioanalyzer, lossis pom qhov pom ntawm Qubit qis dua li ntawm qPCR. Me me dhau qhov kev nthuav dav yog ib qho tshwm sim ib txwm muaj, uas tsis cuam tshuam rau lub tsev qiv ntawv ua ntu zus thiab kev txheeb xyuas cov ntaub ntawv tom ntej.

7. Spikes tshwm hauv qhov txheeb xyuas qhov tseeb ntawm Agilent 2100 Bioanalyzer

Cov tsos ntawm spikes hauv Agilent 2100 Bioanalyzer kuaj pom yog vim qhov tsis sib xws ntawm cov qauv, qhov twg yuav muaj ntau qhov tawg ntawm qee qhov me me, thiab qhov no yuav dhau los ua qhov pom tseeb tom qab PCR txhim kho. Hauv qhov no, nws tau hais qhia kom tsis txhob xaiv qhov loj me, piv txwv li teeb tsa kev faib ua feem rau 94 ° C rau 15 feeb ua kom tsis txhob sib kis, qhov uas faib cov khoom faib me me thiab mloog zoo, thiab homogeneity tuaj yeem txhim kho.

Sau koj cov lus ntawm no thiab xa tuaj rau peb