Kit ɗin ɗakin karatu na TIANSeq (illumina)

A halin yanzu, fasahar jere-jere mai inganci ya dogara ne da fasahar jerawa na gaba. Kamar yadda tsayin karatu na fasahar jerawa na gaba mai iyaka yana da iyaka, dole ne mu raba cikakken jerin tsayin zuwa ƙananan ɗakunan karatu don jere. Dangane da buƙatun gwaje-gwajen jeri daban-daban, galibi muna zaɓar jere ɗaya ko sau biyu. A halin yanzu ana rarraba gutsutsuren DNA na ɗakin karatu na jere na gaba gaba a cikin kewayon 200-800 bp.

a) DNA ba shi da inganci kuma yana ƙunshe da masu hanawa. Yi amfani da samfuran DNA masu inganci don gujewa hana ayyukan enzyme.

b) Adadin samfurin DNA bai isa ba lokacin amfani da hanyar da babu PCR don gina ɗakin karatu na DNA. Lokacin da shigarwar DNA mai rarrabuwa ta wuce 50 ng, ana iya aiwatar da aikin da PCR ba tare da izini ba yayin aikin ginin ɗakin karatu. Idan lambar kwafin ɗakin karatu ta yi ƙasa sosai don a jera ta kai tsaye, PCR za a iya haɓaka ɗakin karatun DNA bayan haɗin adaftar.

c) Gurɓataccen RNA yana haifar da rashin daidaituwa na ƙididdigar DNA na farko RNA na iya kasancewa a cikin tsarin tsarkakewa na kwayar halittar DNA, wanda na iya haifar da ƙididdigar DNA mara inganci da isasshen lodin DNA yayin ginin ɗakin karatu. Ana iya cire RNA ta hanyar magani tare da RNase.

A-1

a) Ƙananan gutsutsure (60 bp-120 bp) ya bayyana Ƙananan gutsutsuren galibi gutsutsuren adaftan ne ko dimers waɗanda masu adaftar suka kafa. Tsarkakewa tare da begen magnetic AMPure XP na Agencourt zai iya cire waɗannan gutsutsuren adaftar yadda yakamata kuma tabbatar da ingancin jerin abubuwa.

b) Manyan gutsuttsura suna bayyana a cikin ɗakin karatu bayan haɓaka PCR Girman guntun DNA na ɗakin karatu zai karu da 120 bp bayan adaftan ya haɗa. Idan gutsurin DNA ya ƙaru da fiye da bp 120 bayan haɗin adaftan, yana iya haifar da haɓaka ɓataccen ɓarna na ƙara girman PCR. Rage adadin hawan keke na PCR na iya hana lamarin.

c) Girman al'ada na gutsutsuren DNA na ɗakin karatu bayan haɗaɗɗen adaftar Tsawon adaftan a cikin wannan kit ɗin shine 60 bp. Lokacin da aka haɗa ƙarshen gutsattsarin guntu ɗin zuwa masu daidaitawa, tsayin zai karu da 120 bp kawai. Lokacin amfani da adaftar banda wanda wannan kit ɗin ya bayar, tuntuɓi mai siyarwa don samar da bayanai masu dacewa kamar tsayin adaftar. Da fatan za a tabbatar cewa aikin gwaji da aiki sun bi matakan da aka bayyana a cikin littafin.

d) Girman guntun DNA mara daidaituwa kafin haɗaɗɗen adaftan Dalilin wannan matsalar na iya zama sanadiyyar halayen da ba daidai ba yayin rarrabuwa na DNA. Ya kamata a yi amfani da lokutan amsawa daban -daban don shigarwar DNA daban -daban. Idan shigarwar DNA ya fi 10 ng, muna ba da shawarar zaɓar lokacin amsawa na mintina 12 a matsayin lokacin farawa don haɓakawa, kuma girman guntun da aka samar a wannan lokacin galibi yana cikin kewayon 300-500 bp. Masu amfani za su iya ƙaruwa ko rage tsawon gutsutsuren DNA na mintuna 2-4 gwargwadon buƙatun nasu don haɓaka gutsutsuren DNA tare da girman da ake buƙata.

A-2

a) Ba a inganta lokacin rarrabuwa Idan ɓataccen DNA ya yi ƙanƙanta ko ya yi yawa, da fatan za a koma zuwa Sharuɗɗan Zaɓin Lokacin Tsagewar da aka bayar a cikin umarnin don ƙayyade lokacin amsawa, da amfani da wannan lokacin azaman iko, bugu da ƙari kafa wani tsarin amsawa don tsawaita ko rage minti 3 don yin madaidaicin daidaituwa akan lokacin rarrabuwa.

A-3

Rarraba girman mahaifa na DNA bayan maganin rarrabuwa

a) Hanyar narkar da ba daidai ba na reagent na rarrabuwa, ko reagent ɗin ba a haɗe shi gaba ɗaya bayan narkewa. Narke 5 -Fragmentation Enzyme Mix reagent akan kankara. Da zarar narke, haɗa reagent a ko'ina ta danna maɓallin bututu a hankali. Kada ku karkatar da reagent!

b) Samfurin shigarwar DNA ya ƙunshi EDTA ko wasu masu gurɓataccen Ruwa na ions gishiri da wakilan chelating a cikin matakin tsarkakewar DNA yana da mahimmanci musamman don nasarar gwajin. Idan an narkar da DNA a cikin 1 × TE, yi amfani da hanyar da aka bayar a cikin umarnin don yin rarrabuwa. Idan maida hankali na EDTA a cikin maganin ba shi da tabbas, ana ba da shawarar a tsarkake DNA kuma a narkar da shi a cikin ruwa mai ɗimbin yawa don ɗaukar mataki na gaba.

c) Ƙididdigar DNA ta farko da ba daidai ba Girman DNA mai rarrafe yana da alaƙa da adadin shigarwar DNA. Kafin maganin rarrabuwa, ƙididdigar DNA daidai ta amfani da Qubit, Picogreen da sauran hanyoyin yana da mahimmanci don ƙayyade ainihin adadin DNA a cikin tsarin amsawa.

 d) Shirye -shiryen tsarin mayar da martani baya bin umarnin Dole ne a aiwatar da tsarin rabe -raben rarrabuwa a kan kankara bisa ga umarnin. Don tabbatar da mafi kyawun sakamako, yakamata a sanya duk abubuwan haɗin kan kan kankara kuma yakamata a aiwatar da shirye -shiryen tsarin amsawa bayan cikakken sanyaya. Bayan an gama shirye -shiryen, da fatan za a yi birgima ko bututu don haɗuwa sosai. Kada ku yi vortex!

1. Hanyar da ba ta dace ba (vortex, oscillation tashin hankali, da sauransu) za ta haifar da rarraba ɓoyayyen ɓoyayyen ɗakin karatu (kamar yadda aka nuna a adadi mai zuwa), ta haka yana shafar ingancin ɗakin karatu. Don haka, lokacin da ake shirya maganin gurɓataccen ɓarna, don Allah a hankali a ɗora sama da ƙasa don haɗawa, ko amfani da yatsan yatsa don jujjuyawa da daidaita daidai. Yi hankali kada ku gauraya da vortex.

2. Dole ne a yi amfani da babban tsarkin DNA don ginin ɗakin karatu

Kyakkyawan amincin DNA: Ƙungiyar electrophoresis ta fi 30 kb, ba tare da wutsiya ba

2 OD260/230:> 1.5

OD260/280: 1.7-1.9

3. Adadin shigar DNA ya zama daidai An ba da shawarar yin amfani da hanyoyin Qubit da PicoGreen don auna DNA, maimakon Nanodrop.

4. Dole ne a ƙaddara abubuwan da ke cikin EDTA a cikin maganin DNA EDTA tana da babban tasiri a kan rabe -raben rarrabuwa. Idan abun cikin EDTA ya yi yawa, ana buƙatar yin tsarkakewar DNA kafin gwajin na gaba.

5. Dole ne a shirya maganin rarrabuwa akan kankara Tsarin rarrabuwa yana da mahimmanci ga zafin zafin da lokaci (musamman bayan ƙara haɓakawa). Domin tabbatar da daidaiton lokacin amsawa, da fatan a shirya tsarin amsawa akan kankara.

6. Dole ne lokacin amsawar rabe -raben ya zama daidai Lokacin amsawar matakin gutsuttsura zai shafi girman samfuran gutsutsuren, ta haka zai shafi girman rarraba gutsutsuren DNA a cikin ɗakin karatu.

1. Wane irin samfurin ne ya dace da wannan kit ɗin?

Samfurin samfurin da ya dace na wannan kit ɗin na iya zama cikakken RNA ko mRNA mai tsarkakewa tare da kyakkyawan amincin RNA. Idan ana amfani da jimlar RNA don gina ɗakin karatu, ana ba da shawarar yin amfani da kitsewar rRNA (Cat#4992363/4992364/4992391) don cire rRNA da farko.

2. Za a iya amfani da samfuran FFPE don gina ɗakin karatu tare da wannan kayan?

MRNA da ke cikin samfuran FFPE za a ƙasƙantar da su har zuwa wani matsayi, tare da rashin aminci na dangi. Lokacin amfani da wannan kit ɗin don ginin ɗakin karatu, ana ba da shawarar haɓaka lokacin rarrabuwa (gajarta lokacin rarrabuwa ko rashin yin rarrabuwa).

3. Yin amfani da matakin zaɓin girman da aka bayar a cikin littafin samfurin, menene zai iya sa ɓangaren da aka saka ya zama ɗan karkacewa?

Za'a yi zaɓin girman daidai gwargwadon matakin zaɓin girman a cikin wannan littafin samfur. Idan akwai karkacewa, dalilin na iya kasancewa beads magnetic ba a daidaita su da zafin jiki na ɗaki ko kuma ba a gama gauraya su ba, bututu bai yi daidai ba ko kuma ruwan ya kasance a cikin ƙima. Ana ba da shawarar yin amfani da tukwici tare da ƙarancin talla don gwajin.

4. Zaɓin adaftan a ginin ɗakin karatu

Kit ɗin ginin ɗakin karatu bai ƙunshi reagent adaftan ba, kuma ana ba da shawarar yin amfani da wannan kit ɗin tare da TIANSeq Adaftar Index (Illumina) (4992641/4992642/4992378).

5. QC na ɗakin karatu

Gano adadi mai yawa na ɗakin karatu: Ana amfani da Qubit da qPCR don ƙayyade taro mai yawa da haɓakar ɗakin ɗakin karatu bi da bi. Anyi aikin daidai gwargwadon littafin samfurin. Haɓaka ɗakin ɗakin karatu gabaɗaya zai cika buƙatun tsarin NGS. Gano kewayon rarraba ɗakin karatu: Amfani da Agilent 2100 Bioanalyzer don gano kewayon rarraba ɗakin karatu.

6. Zaɓin lambar zagayowar ƙara girma

Dangane da umarnin, adadin zagayowar PCR shine 6-12, kuma yakamata a zaɓi adadin abubuwan hawan PCR da ake buƙata gwargwadon shigarwar samfurin. A cikin ɗakunan karatu masu ɗimbin yawa, fiye da haɓaka yawanci yana faruwa a cikin digiri daban-daban, wanda ke bayyana ta ɗan ƙaramin girma mafi girma bayan ƙimar maƙasudi a cikin gano Agilent 2100 Bioanalyzer, ko gano ƙimar Qubit ya yi ƙasa da na qPCR. Ƙarfi akan ƙarawa abu ne na al'ada, wanda baya shafar jerin ɗakunan karatu da nazarin bayanan da ke gaba.

7. Spikes yana bayyana a bayanin gano Agilent 2100 Bioanalyzer

Bayyanar spikes a cikin Agilent 2100 Bioanalyzer gano shine saboda rarrabuwar samfuran, inda za a sami ƙarin gutsuttsura a cikin wani girman, kuma wannan zai zama a bayyane bayan wadatar PCR. A wannan yanayin, ana ba da shawarar kada a yi zaɓin girman, watau saita yanayin rarrabuwa zuwa 94 ° C na mintina 15 da aka ƙone, inda raunin gutsuttsuran ƙarami ne kuma mai da hankali, kuma ana iya inganta daidaituwa.