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RNAÀÇ 5¡¯-¸»´Ü¿¡ Cap (Cap 0)À» ºÎ°¡ÇÏ´Â È¿¼Ò

Vaccinia Capping Enzyme

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Takara
2460A
Vaccinia Capping Enzyme
°ü·ÃÇмú ±¸¸ÅÇϱâ
500 U
220,800¿ø 
276,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
2460A_DS.v2208Da.pdf
Takara
2460B
Vaccinia Capping Enzyme
°ü·ÃÇмú ±¸¸ÅÇϱâ
2,000 U (2460A x 4)
992,000¿ø 

  • 5’-triphosphate RNA¿¡ 7-methylguanylate(m7G) cap ºÎ¿© (Cap 0 ±¸Á¶)
Á¦Ç°¼³¸í
Vaccinia Capping Enzyme (VCE)Àº vaccinia ¹ÙÀÌ·¯½º À¯·¡ÀÇ capping enzymeÀ¸·Î RNA (5’-triphosphate)ÀÇ 5’ ¸»´Ü¿¡ 7-methylguanylate(m7G) cap ±¸Á¶¸¦ ºÎ°¡ÇÏ´Â È¿¼ÒÀÌ´Ù. ÁøÇÙ»ý¹°¿¡¼­ ÀÌ·¯ÇÑ Cap ±¸Á¶´Â mRNAÀÇ stabilization, nuclear export ¹× translation¿¡¼­ ¸Å¿ì Áß¿äÇÑ ¿ªÇÒÀ» ÇÑ´Ù.
ÀÌ È¿¼Ò´Â 2°³ÀÇ subunit (D1, D12)À¸·Î ÀÌ·ç¾îÁ® RNA triphosphatase, guanylyltransferase, guanine methyltransferaseÀÇ ¼¼°¡Áö È¿¼Ò È°¼ºÀ» ³ªÅ¸³½´Ù. ÀÌ Á¦Ç°Àº È¿¼Ò¸¦ È°¼ºÈ­½ÃÅ°±â À§ÇÑ ¸ðµç ±¸¼ºÇ° (10×Capping Buffer, S-adenosylmethionine (SAM) ¹× GTP)À» Æ÷ÇÔÇÏ°í ÀÖ¾î, in vitro »ó¿¡¼­ Cap 0 ±¸Á¶¸¦ °¡Áø RNA¸¦ Á¦ÀÛÇϴµ¥ »ç¿ëÇÒ ¼ö ÀÖ´Ù.
¶ÇÇÑ ÀÌ È¿¼Ò¸¦ mRNA Cap 2'-O-Methyltransferase (Code 2470A)¿Í °°ÀÌ »ç¿ëÇϸé Cap 0 ±¸Á¶¸¦ °¡Áø RNAÀÇ Ã¹¹ø° nucleotide 2’-O ºÎÀ§¸¦ ¸ÞÆ¿È­ÇÏ¿© Cap 1 ±¸Á¶¸¦ °¡Áø RNA¸¦ one-stepÀ¸·Î Á¦ÀÛÇÒ ¼ö ÀÖ´Ù (»ç¿ëÀÚ ¸Þ´º¾ó protocol-2 Âü°í).
¿ëµµ
- in vitro trnascription (IVT)µîÀ¸·Î ÇÕ¼ºµÈ RNAÀÇ 5’-capping (Cap 0) ±¸Á¶ ºÎ°¡
(Takara IVTpro™ T7 mRNA Synthesis Kit (Code 6144)¿Í °°ÀÌ »ç¿ë °¡´É)
- mRNA Cap 2'-O-Methyltransferase (Code 2470A)¿Í °°ÀÌ »ç¿ëÇÏ¿© one-stepÀ¸·Î RNAÀÇ 5’-cap (Cap 1) ±¸Á¶ ºÎ°¡
- mRNAÀÇ 5’ ¸»´Ü labelling
³»¿ë

500 U

 

Vaccinia Capping Enzyme (10 U/§¡)

100 §¡

 

10× Capping Buffer

100 §¡

 

S-adenosylmethionine (SAM) (32 mM)

50 §¡

 

GTP (10 mM)

º¸Á¸ -20¡É
³óµµ 10 U/§¡
±â¿ø
Escherichia coli carrying plasmids containing the genes for vaccinia virus capping enzyme
ÀϹÝÀûÀÎ ¼ºÁú
- Áú·®: ¾à 98 kDa ¹× 33 kDa (heterodimer)
È°¼ºÀÇ Á¤ÀÇ
37¡É¿¡¼­ 1½Ã°£ µ¿¾È 10 pmolÀÇ [¥á-32P]GTP¸¦ RNA transcript¿¡ ºÎ°¡ÇÏ´Â È¿¼Ò·®À» 1 UÀ¸·Î ÇÑ´Ù.
È°¼º ÃøÁ¤¿ë ¹ÝÀÀÁ¦ Á¶¼º

 

Capping Buffer

1 μM

 

GTP

10 μCi/§¢

 

[α-32P]GTP

0.67 mM

 

SAM

2.5 §¶/50 §¡

 

100 nt RNA

»ç¿ë»óÀÇ ÁÖÀÇ
- È¿¼ÒÀÇ °­ÇÑ ±³¹ÝÀº »ï°¡ÇÑ´Ù.
- Template RNA ¹× ½ÇÇè ±â±¸¿¡ RNase°¡ ¿À¿°µÈ °æ¿ì RNAÀÇ yield°¡ ³·¾ÆÁö°Å³ª fragmentation µÈ´Ù. ¹ÝÀÀ¿¡ »ç¿ëÇÏ´Â Æ©ºê, micropipette tip µîÀ» RNA ½ÇÇè Àü¿ëÀ¸·Î »ç¿ëÇÏ°í ÀÏȸ¿ë Àå°©À» »ç¿ëÇÏ¿© RNaseÀÇ ¿À¿°ÀÌ ÀϾÁö ¾Êµµ·Ï ÁÖÀÇÇÑ´Ù.
°ü·ÃÁ¦Ç°
- Cap 0 ±¸Á¶¸¦ °¡Áø RNA¸¦ Cap 1À¸·Î ¸¸µå´Â È¿¼Ò mRNA Cap 2'-O-Methyltransferase (Code 2470A)
- °íÇ°Áú °í¼öÀ² in vitro transcription ½Ã½ºÅÛ Takara IVTpro™ mRNA Synthesis System (Code 6141)
- Transfection ½Ã¾à Àü¹® ºê·£µå MirusÀÇ mRNA Àü¿ë Transfection ½Ã¾à TransIT®-mRNA Transfection Kit (Code MIR 2250)
- ½ÇÇè ±â±â¿¡¼­ RNase ¿À¿° Á¦°Å RNase-OFF® (RNase Contamination Á¦°Å ¿ë¾×) (Code 9037)

Keyword : VCE

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