prou
Nā huahana
HCP0036A HCP0036A kiʻi i hōʻike ʻia i loko o ka vitro.
  • Hiki i ka hua T7 in vitro transcription reagent (Thermotable) HCP0036A

Hiki i ka hua T7 in vitro transcription reagent (Thermotable)


Helu popoki:HCP0036A

Pūʻolo: 50T

Hiki ke hoʻopaʻa ʻia i loko o ka vitro transcription T7 in vitro transcription reagent kit (Thermotable) i nā wela kiʻekiʻe a hiki i 50°C.

Hōʻike huahana

ʻikepili huahana

Hiki ke hoʻopaʻa ʻia i loko o ka vitro transcription T7 in vitro transcription reagent kit (Thermotable) i nā wela kiʻekiʻe a hiki i 50°C.Hiki i ka pahu ke hoʻohui i nā transcripts RNA kiʻekiʻe me ka hoʻohana ʻana i ka DNA stranded linear ʻelua i loaʻa i ka mea hoʻolaha T7 ma ke ʻano he template a me nā NTP ma ke ʻano he substrate.He kūpono ke kit no ka hoʻohui ʻana i nā nucleotides i hoʻololi ʻia e loaʻa ai ka biotin labeled, dye labeled a radiolabeled RNA.Hiki ke hoʻohana ʻia ka pahu i ka synthesis o ka co-transcriptionally capping mRNAs me nā analogue cap.Aia i loko o ka pahu nā nucleotides N1-Me-pUTP i hoʻololi ʻia ma ke ʻano he ʻokoʻa.

Hāʻawi kēlā me kēia hopena maʻamau i ka 150-200 μg o RNA mai 1μg DNA template.Hiki ke hoʻonui ʻia ka nui o ka hopena e hoʻohua i ka milligram-level RNA e like me ka mea e pono ai.

ʻO ka RNA synthesized mai ka kit he kūpono ia no nā noi he nui e pili ana i ka hoʻolālā RNA a me nā haʻawina hana, ribozyme biochemistry, RNase palekana assays a me hybridization-based blots, anti-sense RNA a me RNAi hoʻokolohua.ʻO ka RNA synthesized mai ka kit ua kūpono hoʻi no ka hana ʻana i ka hana enzymatic o ka RNA i hoʻopaʻa ʻia e ka vaccinia capping enzyme a me 2'-O-Methyltransferase a hoʻopili ʻia me ka Poly (A) polymerase e hoʻomaikaʻi i ka paʻa a me ka mākaukau unuhi o RNA i hoʻohana ʻia no ka unuhi in vitro. , hoʻololi, a me ka microinjection.


  • Mua:
  • Aʻe:

  • Nā ʻāpana

    ʻāpana

    Hoʻopaʻa

    Volume

    ATP

    100 mM

    100 μL

    CTP

    100 mM

    100 μL

    GTP

    100 mM

    100 μL

    UTP

    100 mM

    100 μL

    N1-Me-pUTP

    100 mM

    100 μL

    10 × Hoʻonui i ka IVT Buffer A

    /

    100 μL

    Hui Enzyme (Thermotable)

    /

    100 μL

     

    Nā kūlana mālama

    ʻO ka halihali ma lalo o 0°C a me ka mālama ʻana ma -25~-15°C.

     

    Kūkākūkā

    • Hoʻomākaukau hoʻohālike DNA

    Hiki ke hoʻohana ʻia ka DNA plasmid linearized, nā huahana PCR a i ʻole synthetic DNA oligonucleotides ma ke ʻano he hoʻohālike no ka transcription in vitro me ka pahu.

    Plasmid nā laʻana: Pono i loko o ka plasmid linearized kahi wahi hoʻolaha T7 ma ke ʻano he hoʻohālike DNA.Hoʻopili ka maikaʻi o ka plasmid linearized i ka hua a me ka pono o RNA.He mea koʻikoʻi no ka hoʻohana maikaʻi ʻana i ke kit no ka mea ʻaʻole i hoʻopau pono ʻia ka plasmid circular.Loaʻa ka hua unuhi kiʻekiʻe loa me ka maʻemaʻe kiʻekiʻe loa.No ka hana ʻana i ka RNA transcript o kahi lōʻihi i wehewehe ʻia, pono e hoʻokaʻawale ʻia ka plasmid DNA me kahi enzyme kaohi e hoʻohua ai i nā hopena blunt a i ʻole 5'-overhangs.ʻO ka plasmid linearized i hoʻomaʻemaʻe ʻia e nā ʻano hana ʻoihana hiki ke kūʻokoʻa mai ka hoʻohaumia ʻana iā RNase, protein, RNA a me nā paʻakai.

     Nā laʻana PCR: Hiki ke kope ʻia nā huahana PCR i loaʻa i ka T7 RNA polymerase promoter ma ke ʻano kūpono.E loaʻa nā hua maikaʻi me nā huahana PCR i hoʻomaʻemaʻe ʻia, ʻoiai hiki ke hoʻohana pololei ʻia nā huila PCR.

    ʻO ka mea hoʻoheheʻe DNA ʻoligonaucleotides:Hiki ke unuhi ʻia nā oligonucleotides DNA synthetic, ʻo ia hoʻi ka pae ʻelua a i ʻole ka hapa nui o ka pae hoʻokahi me ke kaʻina hoʻolaha T7 kaulua.

    RNA Synthesis Protocols

    ʻO ka hoʻohana ʻana i nā mīkina lima a me ka hoʻohana ʻana i nā paipu nuclease-free a me nā reagents e pale aku i ka haumia RNase.Pono e hōʻuluʻulu ʻia nā pane o ka leo liʻiliʻi i loko o nā paipu microfuge nuclease-free a i ʻole nā ​​paipu PCR strip.

    ʻO RNA Synthesis maʻamau

    1. E hoʻoheheʻe i nā mea pono kit, hui a me ka pulse-spin i ka microfuge e hōʻiliʻili i nā hopena i lalo o nā paipu.E hoomau i ka hau.

    2. Inā ʻoe e hoʻolālā e holo i nā hopena he nui, ʻoi aku ka maʻalahi o ka hoʻomākaukau ʻana i kahi master mix ma ka hoʻohui ʻana i nā helu like o ka 10 × Hi-Yield IVT Buffer a me ʻehā ribonucleotide (NTP).E hoʻohana i 10 µl master mix no kēlā me kēia pane.

    3. E hōʻuluʻulu i ka hopena ma ka lumi wela ma ke ʻano penei:

    Reagent

    Ka nui

    Wai nuclease-free

    X μL

    10 × Hoʻonui i ka IVT Buffer A

    2 μL

    ATP/CTP/GTP/UTP *(100 mM kēlā me kēia)

    2 μL kēlā me kēia (10 mM kēlā me kēia Hope)

    DNA template

    Y μL (1 μg)

    Hui Enzyme (Thermotable)

    2 μL

    Ka nui o ka nui o ka pane

    20 μL

    * No ka hōʻemi ʻana i ka immunogenicity o ka huahana, hiki ke hoʻololi ʻia ka UTP e N1-Me-pUTP ma ka hopena hope loa.

    4. E hui pono, pulse-spin i ka microfuge.Incubate ma 37°C no 2 hola, a i ʻole 50°C no 1 hola inā makemake ʻia ka hopena wela kiʻekiʻe.

    5. DNA Digestion: No ka wehe ʻana i ka DNA template, e hoʻohui i 10U o DNase I i kēlā me kēia 20 μL pane, hui maikaʻi a hoʻomoʻa ma 37 ℃ no 30 mau minuke.

     

    Hoʻopili ʻia ʻo RNA Synthesis

    Kūlike like me ka RNA synthesis maʻamau, koe wale no ka pae o ka hui ʻana i ka hopena.E hōʻuluʻulu i ka hopena o ka RNA synthesis i hoʻopaʻa ʻia ma ka lumi wela ma ke ʻano penei:

    Reagent

    Ka nui

    Wai nuclease-free

    X μL

    10 × Hoʻonui i ka IVT Buffer A

    2 μL

    ATP/CTP/GTP/UTP *(100 mM kēlā me kēia)

    2 μL kēlā me kēia (10 mM kēlā me kēia Hope)

    Kāpena analog (100 mM)

    1.6 μL

    DNA template

    Y μL (1 μg)

    Hui Enzyme (Thermotable)

    2 μL

    Ka nui o ka nui o ka pane

    20 μL

    *** Inā makemake ʻia, hiki ke hoʻohana ʻia ka Cap1(3'OH AG) a me ka cap1(3'OMe AG) i nā analogs ascap.Manaʻo mākou i kā mākou co-transcription reagent Co-capping T7 invitro transcription reagent (pUTP, CAP GAG (3'OMe), pUTP, CAP GAG, N1-Me-pUTP, CAP GAG (3'OMe) a me N1-Me-pUTP, CAP GAG.

     

    mRNA hoomaemae

    • Phenol: Chloroform Extraction a me Ethanol Ka ua

    No ka wehe ʻana i nā protein a me ka hapa nui o nā nucleotides manuahi, ʻo phenol: chloroform extraction a me ethanol precipitation o RNA transcripts ke ala makemake.

    1. Hoʻoponopono i ka nui o ka hopena i 180 μLby me ka hoʻohui ʻana i 160 μL wai nuclease-free.Hoʻohui i 20 μL o 3M sodium acetate, pH5.2, hui maikaʻi.

    2. E unuhi me ka nui like o ka hui phenol/chloroform 1:1, centrifuge ma 10000 rpm no 5 mau minuke.E hōʻiliʻili i ka pae wai a hoʻololi i ka paipu hou.

    3. Hahai ʻia e nā unuhi ʻelua me ka nui like o ka chloroform.E hōʻiliʻili i ka pae wai a hoʻololi i kahi paipu hou.

    4. Ua ho'okahe 'ia ka RNA me 2 mau puke ethanol.Incubate ma -20 ℃ no ka liʻiliʻi loa 30 minuke, a e ohi i ka pallet ma centrifugation no 15 minuke.E wehe pono i ka supernatant.

    5. E holoi i ka pallet me 150 μL ~ 200 μL anu 70% ethanol.

    6. E hoʻomaloʻo i ka pallet no nā minuke 2 a hoʻomaha hou i ka wai 100 μL ~ 200 μL RNase-noa a i ʻole nā ​​mea pale ʻē aʻe.

     

    • LiCl ua

    He mea maikaʻi ka LiCl precipitation o RNA i ka wehe ʻana i ka hapa nui o nā NTP a me nā enzyme i hui ʻole ʻia.

    1. E hoʻohui i ka nui like o ka solution LiCl (5M), e hui maikaʻi.

    2. Hoʻomoʻa ma -20 ℃ no 30 mau minuke.Centrifuge ma 4 ℃ ma 12000 rpm no 15 mau minuke e pallet RNA.E wehe pono i ka supernatant.

    3. Resin ka pallet ma ka hoʻohui ʻana i 200 μL o precooling 70% ethanol.E wehe pono i ka ethanol.E hana hou i nā ʻanuʻu no 2-3 mau manawa.

    4. Hoʻomaloʻo ka ea i ka pallet no 5 ~ 10 mau minuke a hoʻomaha hou i ka wai 100 μL ~ 200 μL RNase-noa a i ʻole nā ​​mea pale ʻē aʻe.

    • Milo hoʻomaʻemaʻe kolamu

    E hoʻoneʻe nā kolamu spin i nā NTP i hui ʻole ʻia, nā protein a me nā paʻakai.

    • Puri mākēnekihoʻopaʻapaʻa

    Hiki i ka hoʻomaʻemaʻe ʻana i ka bead magnetic ke wehe i nā nucleotides i hui ʻole ʻia, nā protein a me nā paʻakai.

     

    Ka helu o nā huahana pane

    • Ka helu ana e UV hoʻopaʻa māmā: Pono ka hoʻomaʻemaʻe ʻana i nā huahana no ka mea, ʻo nā nucleotides i hoʻohui ʻole ʻia a me ka DNA template i loko o nā hui ʻana e pili ana i ka heluhelu.ʻO ke ana ʻana i ka spectrophotometry UV ma 260 nm hiki ke loaʻa maʻalahi ka ʻike RNA.No ka RNA kaula hoʻokahi, pili ka 1 A260 i ka ʻike RNA o 40 μg/mL.

     Ka helu ana by kalai: Hiki ke hoʻohana ʻia ka helu o nā huahana hopena me ka waiʻona Ribogreen me ka hoʻomaʻemaʻe ʻole ʻana no ka mea ʻaʻole pili nā nucleotides i hoʻohui ʻole ʻia i ka loiloi o nā huahana RNA.

      

    Nā memo

    • Pono e hoʻokō ʻia ka hopena o ka unuhi ʻana ma ke kaiapuni ʻole RNase.Pono ke komo lima lima.ʻO nā ʻōlelo aʻoaʻo, nā paipu a me ka wai e pono ʻole ka nuclease.

    • ʻO 10 mM ka nui loa o ka manaʻo hope loa o nā NTP, a hiki iā ʻoe ke hoʻololi i ka ʻike hope loa o nā NTP e like me ke kūlana maoli.

    • Pono e hoʻomākaukau ʻia ka hui ʻana o ka RNA synthesis reaction ma ka lumi wela, no ka mea, hiki i ka DNA ke hoʻoheheʻe i mua o ka spermidine ma 4°C.

    • E emi ana ka hua o ka loa'a pono 'ana ke ho'onohonoho pono 'ole ka DNA template.

    • Hiki ke hoʻonui a i lalo ka hui ʻana.

    • E hoʻohui i ka pahu a hiki i ka hoʻoheheʻe ʻia ʻana o nā mea hiki ʻole ke hoʻoheheʻe ʻia ma mua o ka hoʻohana ʻana, inā ʻike ʻia he mau mea hiki ʻole ke hoʻoheheʻe ʻia ma hope o ka hoʻoheheʻe ʻana.

    • No nā pane me nā transcripts ʻoi aku ka pōkole ma mua o 300 bp, 4-8 h incubation ma 37 ℃ pono e hāʻawi iā ʻoe i ka hua kiʻekiʻe.

    • Pono ka la'ana DNA i ho'ohana 'ia no ka RNA synthesis transcripts ma'amau i ka T7 promoter sequence a me ka GGG initiation sequence, no ka mea, he ki'eki'e ka pili o GTP T7 RNA polymerase.

    • Pono e loa'a i ka palapala DNA i ho'ohana 'ia no ka mRNA synthesis me ka co-transcription system i ka T7 promoter sequence i ukali ia e ka AGG sequence ho'omaka.

    Hoʻoponopono pilikia

    a) Haʻahaʻa haʻahaʻa o ka piha-length RNA:

    Inā hoʻopuka ka hopena transcription i ka RNA holoʻokoʻa, akā ʻoi aku ka haʻahaʻa o ka hua ma mua o ka mea i manaʻo ʻia, hiki i nā mea haumia i loko o ka template DNA ke keʻakeʻa nei i ka RNA polymerase, a i ʻole ka haʻahaʻa a i ʻole ka pololei o ka ʻike DNA.

    Manaʻo: Manaʻo ʻia ka hoʻomaʻemaʻe hou ʻana i ka template DNA.

     

    b) RNA transcript e hamo ana denaturing Gel:

    Inā ʻike ʻia ka hoʻohaʻahaʻa ʻana o ka RNA ma ka denaturing agarose a i ʻole polyacrylamide gel, ua haumia ka DNA template a i ʻole kaʻina hana hoʻokolohua me RNase.

    Manaʻo: Inā haumia ka DNA plasmid me RNase, e hana i ka phenol/chloroform extraction a me ka ethanol precipitate.E hōʻoia i nā ʻōlelo aʻoaʻo a me nā paipu i hoʻohana ʻia i ka wā hoʻokolohua he RNase-noa.E ʻoluʻolu e ʻaʻahu i ka lole lab, ka maka a me nā mīkina lima hoʻopau.

     

    c) RNA transcript of ʻoi aku ka nui ma mua o ka mea i manaʻo ʻia:

    Inā ʻoi aku ka nui o ka RNA transcript ma mua o ka mea i manaʻo ʻia ma kahi gel denaturing, hiki ke hoʻopau piha ʻia ka DNA plasmid template.ʻO ka hele ʻana o nā hale hana lua ikaika hiki ke hoʻopau ʻole i ka transcript RNA.

    Manaʻo Manaʻo: E nānā i ka laʻana no ka hoʻoheheʻe piha ʻana, inā hoʻopaʻa ʻia ka plasmid undigested, e hana hou i ka hoʻopaʻa ʻana i ka enzyme digestion.E ho'ēmi i nā kūkulu lua o ka laʻana DNA ma ke kahua o ka hoʻolālā kaʻina.

     

    d) RNA transcript o liʻiliʻi nui ma mua o i manaʻo ʻia:

    Inā hōʻike ka hōʻike ʻana i ka gel denaturing i ka loaʻa ʻana o nā kaula liʻiliʻi ma mua o ka nui i manaʻo ʻia, ma muli paha ia o ka hoʻopau mua ʻana e ka polymerase.ʻO kekahi mau kaʻina e like me nā hōʻailona hoʻopau polymerase T7 RNA e hoʻopau koke.

    Manaʻo Manaʻo: ʻO ka hoʻoulu ʻana i ka hopena transcription ma nā haʻahaʻa haʻahaʻa, no ka laʻana ma 30°C, hiki ke hoʻonui i ka hapa o ka transcript lōʻihi piha, akā e hoʻemi ʻia ka hua.No nā mamana GC waiwai, a i ʻole nā ​​hiʻohiʻona me nā hale kiʻekiʻe, hiki i ka incubation ma 42°C ke hoʻomaikaʻi i ka hua o ka transcript lōʻihi.

    E kākau i kāu leka ma aneʻi a hoʻouna mai iā mākou