´Ý±â
TaKaRa
Á¦Ç°¸í/Á¦Ç°ÄÚµå
Å°¿öµå°Ë»ö
°Ë»ö
´Ý±â
  • °í°´Áö¿ø 
  • ¾÷¹«¾È³»¦¢Á¦Ç°¹®ÀÇ
  • ÀüÈ­¹øÈ£¦¢02-2081-2510
  • Email     ¦¢support@takara.co.kr
  • ´ëÀüÁö»ç 
  • ¾÷¹«¾È³»¦¢´ëÀü/ÃæûÁö¿ª ÁÖ¹®, Á¦Ç°¹®ÀÇ
  • ÀüÈ­¹øÈ£¦¢042-828-6525
  • Email     ¦¢tkbd@takara.co.kr
  • ¾÷¹«½Ã°£¾È³»
  • [ Æò¡¡¡¡ÀÏ ] 09 : 00 ~ 18 : 00 ¦¢ [ Á¡½É½Ã°£ ] 12 : 00 ~ 13 : 00
  • Å䡤ÀÏ¿äÀÏ, °øÈÞÀÏÀº ÈÞ¹«ÀÔ´Ï´Ù.
Home > ÀüÁ¦Ç°º¸±â > mRNA ¹é½Å Ä¡·áÁ¦ °³¹ß > in vitro transcription > PrimeCap¢â T7 RNA Polymerase (low dsRNA)
dsRNA »ý¼ºÀÌ Àû¾î ÀÓ»óºÐ¾ßÀÇ ¿¬±¸°³¹ß¿¡ ÀûÇÕÇÑ °íÇ°Áú mRNA ÇÕ¼º

PrimeCap¢â T7 RNA Polymerase (low dsRNA)

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Takara
2560A
PrimeCap¢â T7 RNA Polymerase (low dsRNA)
°ü·ÃÇмú ±¸¸ÅÇϱâ
20,000 U
266,400¿ø 
333,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
2560A_DS.v2401Da.pdf

  • IVT (in vitro transcription)¿¡¼­ RNA ÇÕ¼º ½Ã ¹ß»ýÇÏ´Â double stranded RNA (dsRNA) »ý¼ºÀ» Å©°Ô ¾ïÁ¦ÇØ »ýüÅõ¿© ½Ã ¸é¿ª¿ø¼º °¨¼Ò
  • CleanCap¢ç Reagent AG (TriLink »ç)µîÀÇ cap analog¿Í °°ÀÌ »ç¿ëÇØ capping È¿À²ÀÌ ³ôÀº °í¼öÀ² RNA ÇÕ¼º °¡´É
  • ±âÁ¸ Á¦Ç° T7 RNA Polymerase ver.2.0 (Code 2541A)¿¡ ºñÇØ ³»¿­¼ºÀ» ³ô¿© (~52¡É) ´Ù¾çÇÑ Á¶°Ç¿¡¼­ ¹ÝÀÀ °¡´É
Á¦Ç°¼³¸í
PrimeCap¢â T7 RNA Polymerase (low dsRNA)´Â T7 RNA Polymerase*1¸¦ ¾÷±×·¹À̵åÇÏ¿©, ÃÖÀú·Î dsRNA »ý¼ºÀ» ¾ïÁ¦ÇÑ Cap analog dependentÇÑ RNAÇÕ¼ºÀÌ °¡´ÉÇÏ°í, ³»¿­¼º (~52¡É)À» °¡Áöµµ·Ï °³·®µÇ¾ú´Ù. ÀÌ È¿¼Ò¸¦ CleanCap¢ç Reagent AGµîÀÇ cap analog¿Í °°ÀÌ »ç¿ëÇÏ¸é ³ôÀº capping È¿À²°ú °í¼öÀ² mRNA ÇÕ¼ºÀÌ °¡´ÉÇϸç, µ¿½Ã¿¡ dsRNA·Î ÀÎÇØ ¹ß»ýÇÏ´Â ¸é¿ª¿ø¼ºÀ» Å©°Ô ÁÙÀÏ ¼ö ÀÖ´Ù. °íÇ°ÁúÀÇ mRNA¸¦ ´ë·®À¸·Î Á¦Á¶ÇÒ ¼ö Àֱ⠶§¹®¿¡ ¹é½Å°ú °°Àº RNA therapy ºÐ¾ßÀÇ ¿¬±¸ °³¹ß¿¡ ÀûÇÕÇÏ´Ù.

*1 NTP¸¦ ±âÁú·Î double-stranded DNA¿¡¼­ T7 promoterÀÇ downstream ¿µ¿ªÀ» RNA·Î ÇÕ¼º (transcription)ÇÏ´Â È¿¼Ò.

ÀϹÝÀûÀÎ T7 promoter ¼­¿­ ¹× transcription start site (CleanCap¢ç Reagent AG »ç¿ë ½Ã ÁÖÀÇ»çÇ×)
IVT¸¦ À§ÇØ ÇÊ¿äÇÑ template DNAÀÇ ÀϹÝÀûÀÎ T7 promoter ¼­¿­Àº ¾Æ·¡ ±×¸²°ú °°´Ù. ´Ü, ÇÕ¼ºÇÏ°íÀÚ ÇÏ´Â RNA ¿°±â ¼­¿­ÀÇ Æ¯Â¡°ú cap analog »ç¿ë ¿©ºÎ¿¡ µû¶ó templateÀ» ´Ù¸£°Ô ¼³°èÇØ¾ß ÇÑ´Ù.


[CleanCap Reagent AG »ç¿ë½Ã]
Transcription start site (+1)ÀÇ NGG¼­¿­À» AGG·Î ¼³°èÇÏ´Â °ÍÀ» ±ÇÀå

[uncapped RNA¸¦ ÇÕ¼ºÇϰųª ARCA¸¦ »ç¿ëÇÏ´Â °æ¿ì]
Transcription start site (+1)ÀÇ NGG¼­¿­À» GGG·Î ¼³°èÇÏ´Â °ÍÀ» ±ÇÀå


±×¸² 1. FLuc mRNA (¾à 1.9 kb)ÀÇ ÇÕ¼º ¼öÀ² (A), dsRNA »ý¼ºÀ² (B) ¹× dsRNAÀÇ ºñÀ² (C)
Positive Control Template (FLuc) (Takara IVTpro¢â T7 mRNA Synthesis Kit (Code 6144)ÀÇ ±¸¼ºÇ°)À» ÁÖÇüÀ¸·Î T7 RNA Polymerase ver.2.0 (Code 2541A)¿Í º» Á¦Ç°À» »ç¿ëÇØ mRNA ÇÕ¼º À» ºñ±³ÇÑ °á°ú (20 §¡, »ç¿ë¿¹ Âü°í), º» Á¦Ç°ÀÌ mRNA ÇÕ¼º ¼öÀ²ÀÇ ÀúÇÏ ¾øÀÌ dsRNA »ý¼ºÀ²À» 90% ÀÌ»ó °¨¼Ò½ÃÅ°´Â °ÍÀ» È®ÀÎÇÒ ¼ö ÀÖ´Ù.


±×¸² 2. Cap analogÀÇ ³óµµ º° mRNA ÇÕ¼º ¼öÀ² (A), Capping È¿À² (B) ¹× ´Ü¹éÁú ¹ßÇöÀ² (C).
Positive Control Template (FLuc)À» ÁÖÇüÀ¸·Î ´Ù¾çÇÑ ³óµµÀÇ CleanCap Reagent¢ç AG (0, 2, 4, 6, 8 mM)¿Í º» Á¦Ç° ¶Ç´Â T7 RNA Polymerase ver.2.0 (Code 2541A)À¸·Î FLuc mRNA (¾à 1.9 b)¸¦ ÇÕ¼ºÇÏ°í ¼öÀ²À» ÃøÁ¤ÇÏ¿´´Ù. ÀÌÈÄ ÇÕ¼ºµÈ mRNA´Â LC/MS·Î capping È¿À²À» ÃøÁ¤ÇÏ¿´°í, HEK293 cell¿¡ transfectionÇØ FLuc assay·Î ´Ü¹éÁú ¹ßÇö·®À» ÃøÁ¤Çß´Ù. ±× °á°ú, º» Á¦Ç°Àº CleanCap¢ç Reagent AG ³óµµ¿¡ ºñ·ÊÇÏ¿© mRNA ÇÕ¼º ¼öÀ²ÀÌ Áõ°¡ÇÏ¿´°í, Àú³óµµÀÇ CleanCap Reagent AG¿¡¼­µµ ³ôÀº capping È¿À²À» º¸¿´À¸¸ç, ´Ù¾çÇÑ Á¶°Ç¿¡¼­ ´Ü¹éÁú ¹ßÇöÀ²ÀÌ ³ôÀº mRNA¸¦ ÇÕ¼ºÇÏ¿´´Ù.


±×¸² 3. IVT ¹ÝÀÀ½Ã »ý¼ºµÇ´Â dsRNAÀÇ ¿¹
ÀϹÝÀûÀÎ T7 RNA Polymerase´Â RNA ÇÕ¼º °úÁ¤¿¡¼­ ¹ß»ýÇϴ ªÀº ´ÜÆíÀÇ ssRNA ȤÀº RNA ¸»´Ü¿¡ ºñƯÀÌÀûÀ¸·Î ºÎ°¡µÇ´Â Ãß°¡¼­¿­ (¸»´Ü ÀüÀÌ È°¼º)·Î ÀÎÇØ dsRNA°¡ Çü¼ºµÇ°í ÀÌ´Â ¸é¿ª¿ø¼ºÀ¸·Î ÀÛ¿ëÇØ mRNAÀÇ ¾ÈÀü¼º°ú È¿À²À» °¨¼Ò½Ãų ¼ö ÀÖ´Ù.
º¸Á¸: -20 ¡É
³»¿ë

±¸¼ºÇ°

¿ë·®

PrimeCap¢â T7 RNA Polymerase (low dsRNA)

20,000 U

10X T7 RNA Polymerase Buffer

1 §¢

³óµµ: 200 U/§¡
¿ëµµ
- Cap analog¸¦ »ç¿ëÇÑ capped mRNA ÇÕ¼º (co-transcriptional capping)
- Capping enzymeÀ» Àû¿ëÇϱâ À§ÇÑ uncapped RNA ÇÕ¼º (Âü°í; cap analog ´ëºñ 10~40% ³·Àº RNA ¼öÀ²)
±â¿ø
Escherichia coli carrying a plasmid containing the gene for phage T7 RNA polymerase variant
ÀϹÝÀûÀÎ ¼ºÁú
- ºÐÀÚ·®: ¾à 99.8 kDa
- Cofactor: Mg2+
È°¼ºÀÇ Á¤ÀÇ
37 ¡É¿¡¼­ 1½Ã°£ µ¿¾È 1nmol [3H] GMP¸¦ acid-insoluble fraction¿¡ »ý¼ºÇÏ´Â È¿¼ÒÀÇ ¾çÀ» 1 UÀ¸·Î ÇÑ´Ù.
È°¼º ÃøÁ¤¿ë ¹ÝÀÀ¾×ÀÇ Á¶¼º

40 mM

 

Tris-HCl, pH 8.0

8 mM

 

MgCl2

2 mM

 

spermidine)

5 mM

 

DTT

0.4 mM

 

ATP, UTP, and CTP

0.4 mM

 

[3H]GTP

1 §¶/50 §¡

 

pT7-2 DNA

ÁÖÀÇ»çÇ×
1. °úµµÇÑ vortexingÀ» »ï°¡ÇØ ÁÖ¼¼¿ä.
2. Template DNA³ª ¹ÝÀÀ¿¡ »ç¿ëµÇ´Â tube, micro-pipette µîÀÌ RNase¿¡ ¿À¿°µÇ¾î ÀÖ´Â °æ¿ì RNAÀÇ ¼öÀ²ÀÌ ³·¾ÆÁö°Å³ª ´ÜÆíÀÌ ºÐÇØµÉ ¼ö ÀÖ½À´Ï´Ù. RNase free tube ¹× micro-pipetteÀ» »ç¿ëÇÏ°í ÀÏȸ¿ë Àå°©À» »ç¿ëÇØ ÁÖ¼¼¿ä.
3. ÀÏÁ¤ÇÑ ±æÀÌÀÇ RNA ÇÕ¼ºÀ» À§ÇØ ¼±ÇüÈ­µÈ plasmid ȤÀº PCR »ê¹°°°Àº linearized dsDNA¸¦ »ç¿ëÇØ¾ß Çϸç, ¿øÄ¡ ¾ÊÀº ÇüÅÂÀÇ RNA ÇÕ¼ºÀ» ¹æÁöÇϱâ À§ÇØ template DNAÀÇ 3¡¯ ¸»´ÜÀº 5¡¯-protruding ȤÀº blunt ÇüŸ¦ ÃßõÇÕ´Ï´Ù.
4. 10X T7 RNA Polymerase Buffer´Â spermidineÀÌ Æ÷ÇԵǾî, ÇÙ»ê°ú º¹ÇÕü¸¦ Çü¼ºÇØ °æ¿ì¿¡ µû¶ó ºÒ¿ë¼ºÀ¸·Î ħÀüµÇ´Â °æ¿ì°¡ ÀÖ½À´Ï´Ù. µû¶ó¼­ template DNA´Â ¹ÝÀÀ¾× Á¶Á¦ ½Ã enzymeÀ» ³Ö±â ¹Ù·Î Á÷Àü¿¡ ¸¶Áö¸· ¼ø¼­·Î ³Ö´Â °ÍÀ» ±ÇÀåÇÕ´Ï´Ù.
5. ÀÌ Á¦Ç°¿¡´Â cap analog ¹× NTP (ATP, CTP, GTP, UTP)°¡ Æ÷ÇԵǾî ÀÖÁö ¾ÊÀ¸¹Ç·Î, CleanCap¢ç Reagent AG (TriLink)¿Í Ribonucleoside 5£§-Triphosphates (Code 4041 ¿Ü)µîÀ» ÁغñÇØ ÁÖ¼¼¿ä.
6. IVT½Ã RNA ÇÕ¼º ¹ÝÀÀÀ» ÃËÁøÇÏ´Â Pyrophosphatase (inorganic) (Code 2450A)¿Í ÇÕ¼ºµÈ RNAÀÇ ºÐÇظ¦ ¾ïÁ¦ÇÏ´Â Recombinant RNase Inhibitor ver.2.0 (Code 2315A)¸¦ °°ÀÌ »ç¿ëÇÏ´Â °ÍÀ» ±ÇÀåÇÕ´Ï´Ù. ÀÚ¼¼ÇÑ ³»¿ëÀº °¢ Á¦Ç°ÆäÀÌÁö¸¦ Âü°í ¹Ù¶ø´Ï´Ù.
»ç¿ë ¿¹

RNase-free Water

 

X §¡

10X T7 RNA Polymerase Buffer

 

2 §¡

ATP, CTP, GTP, UTP*1

 

°¢ 10 mM

CleanCap Reagent AG*2

 

4 mM

Template DNA

 

0.5 ~ 2 §¶

Recombinant RNase Inhibitor ver.2.0

 

20 U

Pyrophosphatase (inorganic)

 

0.1 U

PrimeCap T7 RNA Polymerase

 

200 U

Total

 

20 §¡


37¡É¿¡¼­ 1~2½Ã°£ ¹ÝÀÀ
*1 Modified NTP¸¦ »ç¿ëÇÏ´Â °æ¿ì ÇØ´çÇÏ´Â NTP¸¦ µ¿·®À¸·Î ´ëüÇÏ¿© »ç¿ë
*2 CleanCap Reagent AG (TriLink BioTechnologies: Code. N-7113-1/5/10)
°ü·ÃÁ¦Ç°
- IVT¿ë Plasmid templateÀ» °£´ÜÇÏ°Ô Á¦ÀÛÇÏ´Â Kit: Cloning Kit for mRNA Template (Code 6143)
- Capping enzyme (uncapped RNA -> cap-0 RNA): Vaccinia Capping Enzyme (Code 2460A)
- Capping enzyme (cap-0 RNA -> cap-1 RNA): mRNA Cap 2-O-Methyltransferase (Code 2470A)
- IVTÀü vectorÀÇ ¼±ÇüÈ­¿¡ »ç¿ëÇÏ´Â Type IIS Á¦ÇÑÈ¿¼Ò: BspQ I (Code 1227A)
- BspQ I Àνļ­¿­ÀÌ ÀÖ´Â IVT template vector: Template Vector (BspQ I) for T7 mRNA Synthesis (Code 6146)
- mRNA ¹ßÇöÀ» Boost½Ã¾àÀÌ Æ÷ÇÔµÈ transfection reagent: TransIT¢ç-mRNA Transfection Kit (Code MIR 2250)

Keyword :

in vitro transcription
>
°íÇ°Áú °í¼öÀ² in vitro transcriptionÀ» À§ÇÑ ..
Takara IVTpro¢â mRNA Synthesis System
>
dsRNA »ý¼ºÀÌ Àû¾î ÀÓ»óºÐ¾ßÀÇ ¿¬±¸°³¹ß¿¡ ÀûÇÕ..
PrimeCap¢â T7 RNA Polymerase (low dsRNA)
>
Poly(A) ÀÌÈÄ Ãß°¡¼­¿­ÀÌ ¾ø´Â scarless mRNA ..
Template Vector (BspQ I) for T7 mRNA Synthesis
>
RNAÀÇ 5¡¯-¸»´Ü¿¡ Cap (Cap 0)À» ºÎ°¡ÇÏ´Â È¿¼Ò
Vaccinia Capping Enzyme
>
RNAÀÇ 5¡¯ ¸»´ÜÀ» Cap 0 ¡æ Cap 1·Î º¯È¯ÇÏ´Â ..
mRNA Cap 2-O-Methyltransferase
>
´Ù¾çÇÑ ¿Âµµ¿¡¼­ ¹ÝÀÀÇÒ ¼ö ÀÖ´Â Capping enzym..
Faustovirus Capping Enzyme (S17)
>
Poly(A) ¹× ¹Ýº¹ ¼­¿­À» Æ÷ÇÔÇÏ´Â À¯ÀüÀÚÀÇ Å¬..
Takara Stable Competent Cells
>
RNA ÇÕ¼º ¼öÀ²ÀÌ Çâ»óµÈ
T7 RNA Polymerase ver.2.0
>
Àú³óµµ DTT¿¡¼­µµ ¾ÈÁ¤ÀûÀÎ RNase inhibitor
Recombinant RNase Inhibitor ver.2.0
>
DNA, RNA ÇÕ¼º ¹ÝÀÀÀ» ÃËÁø
Pyrophosphatase (inorganic)
>
IVT Àû¿ë¿¡ ÃÖÀû, Type IIS Á¦ÇÑÈ¿¼Ò
BspQ I
>
´Ù¾çÇÑ Å©±âÀÇ ssRNA »çÀÌÁî È®ÀÎ
0.5-10 kb ssRNA Ladder Marker