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IVT Àû¿ë¿¡ ÃÖÀû, Type IIS Á¦ÇÑÈ¿¼Ò

BspQ I

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Takara
1227A
BspQ I
°ü·ÃÇмú ±¸¸ÅÇϱâ
500 U
112,000¿ø 
140,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
1227A_DS.v2210Da.pdf
Takara
1227B
BspQ I
°ü·ÃÇмú ±¸¸ÅÇϱâ
2,500 U (1227A x 5)
625,000¿ø 

  • Á¦ÇÑÈ¿¼Ò Àνļ­¿­ ÀÏÁ¤ ¿°±â ¹Ù±ù DNA¸¦ Àý´ÜÇÏ´Â Type IIS Á¦ÇÑÈ¿¼Ò (Isochizomer)
  • ºñ´ëĪÀÇ ÀÌÁß°¡´Ú DNA¼­¿­ ÀνÄ
  • Translation È¿À²À» ³ôÀ̱â À§ÇØ in vitro transcription½Ã PolyA ¼­¿­ ÀÌ¿Ü¿¡ ºÒÇÊ¿äÇÑ ¿°±â°¡ ¾ø´Â ÁÖÇü DNA¸¦ Á¦ÀÛÇÒ ¶§ È°¿ë
Á¦Ç°¼³¸í
BspQ IÀº ºñ´ëĪÀÇ Æ¯Á¤ ÀÌÁß°¡´Ú DNA¼­¿­À» ÀνÄÇÏ¿© ÀÏÁ¤ ¿°±â ¹Ù±ùÀÇ DNA¸¦ Àý´ÜÇÏ´Â Type IIS Á¦ÇÑÈ¿¼ÒÀÌ´Ù (Isochizomer). in vitro transcription½Ã, mRNA ÇÕ¼ºÀ» À§ÇÑ ÁÖÇü plasmid¿¡ BspQ I Àνļ­¿­À» Ãß°¡ÇÑ ÈÄ, BspQ IÀ» ó¸®Çϸé PolyA ¼­¿­ ÀÌ¿Ü¿¡ ºÒÇÊ¿äÇÑ ¿°±â°¡ ¾ø´Â mRNA¸¦ ÇÕ¼ºÇÒ ¼ö ÀÖ´Ù.*1
*1 mRNA ÇÕ¼ºÀ» À§ÇÑ ÁÖÇü DNA¸¦ Á¦ÀÛÇÒ ¶§ PolyA ¼­¿­ µÚ¿¡ Ãß°¡ ¿°±â°¡ ³²´Â °æ¿ì mRNAÀÇ translation È¿À²ÀÌ ÀúÇϵǹǷΠ¿°±â°¡ ³²Áö ¾Êµµ·Ï ¼³°èÇÏ´Â °ÍÀÌ Áß¿äÇÏ´Ù (±×¸² 1).


±×¸² 1. Poly A ¼­¿­ µÚ¿¡ Ãß°¡µÇ´Â ¿©ºÐÀÇ ¿°±â ¼ö¿¡ µû¸¥ mRNA translation È¿À² ºñ±³
3 Á¾·ùÀÇ Á¦ÇÑÈ¿¼Ò (BamH I, Hind III, BspQ I)·Î ¼±ÇüÈ­µÈ IVT¿ë plasmid·Î mRNA ÇÕ¼º ÈÄ TransIT®-mRNA Transfection Reagent (Code MIR 2250)¸¦ »ç¿ëÇØ HEK293T ¼¼Æ÷¿¡ transfection ÇÏ¿´´Ù. ±× °á°ú, FLuc positive control°ú ºñ±³Çؼ­ 23°³ÀÇ Ãß°¡ ¿°±â°¡ ³²¾ÆÀÖ´Â mRNA´Â translation È¿À²ÀÌ Å©°Ô ÀúÇϵǾú´Ù.
¹Ý¸é¿¡ 5°³ ÀÌÇÏÀÇ ¿°±â°¡ ³²Àº °æ¿ì ¸ðµÎ ºñ½ÁÇÑ translation È¿À²À» ³ªÅ¸³ÂÁö¸¸, ¿©ºÐÀÇ ¿°±â°¡ ÀûÀ»¼ö·Ï mRNAÀÇ translation È¿À²ÀÌ ´õ ³ôÀº °æÇâÀ» º¸¿´´Ù. Ãß°¡ ¿°±â°¡ ¾øÀ» ¶§ translation È¿À²ÀÌ °¡Àå ³ô±â¿¡ mRNA ÇÕ¼ºÀ» ¸ñÀûÀ¸·Î ÇÏ´Â ÁÖÇü Á¦ÀÛ ½Ã BspQ I°ú °°Àº Type IIS Á¦ÇÑ È¿¼ÒÀÇ »ç¿ëÀ» ±ÇÀåÇÑ´Ù.
¡Ø Hind IIIÁ¦ÇÑÈ¿¼Ò¸¦ »ç¿ëÇØ 3°³ ÀÌÇÏÀÇ ÀÜ¿© ¿°±â°¡ ³²´Â °æ¿ì, translation È¿À²ÀÌ ¼ÒÆø °¨¼Ò µÇ¾úÁö¸¸ ¿©ºÐ ¿°±â°¡ ¾ø´Â °æ¿ì¿Í Å« Â÷À̸¦ º¸ÀÌÁö´Â ¾Ê´Â´Ù.
¿ëµµ
mRNA ÇÕ¼º (in vitro transcription)¿¡ »ç¿ëµÇ´Â ÁÖÇü plasmidÀÇ ¼±ÇüÈ­
³»¿ë

BspQ I (10 U/§¡)

500 U

10x BspQ I Buffer*

1 §¢

* º» ¹öÆÛ¿¡´Â BSA°¡ Æ÷ÇԵǾî ÀÖÁö ¾Ê´Ù.
º¸Á¸ -20¡É
±â¿ø
Escherichia coli carrying the plasmid containing the gene for BspQ I
ÀϹÝÀûÀÎ ¹ÝÀÀ Á¶°Ç
¹ÝÀÀ ¿Âµµ
50¡É*
*ÁÖÀÇ: 37¡É¿¡ ¹ÝÀÀ ½Ã 50¡Éº¸´Ù Àý´Ü È¿À²ÀÌ 60% Á¤µµ·Î ³·¾ÆÁø´Ù.
ÁÖÀÇ»çÇ×
- È¿¼ÒÀÇ °­ÇÑ ±³¹ÝÀº »ï°¡ÇÑ´Ù.
¿­ ½ÇÈ° Á¶°Ç
70¡É, 20ºÐ
¸ÞÆ¿È­ÀÇ ¿µÇâ
dam methylation, dcm methylation¹× CpG methylationÀÇ ¿µÇâÀ» ¹ÞÁö ¾Ê´Â´Ù.
Star È°¼º
Àå½Ã°£ (e.g. 4½Ã°£ ÀÌ»ó) ¹ÝÀÀÇÒ ¶§, star È°¼ºÀÌ »ý±æ ¼ö ÀÖ´Ù.
°ü·ÃÁ¦Ç°
- Çѹø¿¡ 200 §¶ mRNA ÇÕ¼º, °í¼öÀ² in vitro transcription: Takara IVTpro™ T7 mRNA Synthesis Kit (Code 6144)*
- Uncapped RNAÀÇ 5’ ¸»´Ü¿¡ Cap 0 ±¸Á¶¸¦ ºÎ¿©ÇÏ´Â È¿¼Ò Vaccinia Capping Enzyme (Code 2460A)
- Cap 0 ±¸Á¶¸¦ °¡Áø RNA¸¦ Cap 1À¸·Î ¸¸µå´Â È¿¼Ò mRNA Cap 2'-O-Methyltransferase (Code 2470A)
- mRNA Àü¿ë Transfection ½Ã¾à, TransIT®-mRNA Transfection Kit (Code MIR 2250)
- Àú³óµµ DTT¿¡¼­µµ ¾ÈÁ¤ÀûÀÎ ¾÷±×·¹À̵å RNase inhibitor: Recombinant RNase Inhibitor ver.2.0 (Code 2315A)
- RNA ÇÕ¼º ¼öÀ²ÀÌ Çâ»óµÈ Ver.2 T7 RNA Polymerase ver.2.0 (Code 2541A)
- RNA, DNA ÇÕ¼º ½Ã PPi¸¦ Á¦°ÅÇÏ¿© ¼öÀ²À» ³ôÀ̴ Pyrophosphatase (inorganic) (Code 2450A)
- BspQ I°ú °°ÀÌ »ç¿ëÇÒ ¼ö ÀÖ´Â IVT¿ë Template Vector: Template Vector (BspQ I) for T7 mRNA Synthesis (Code 6146)
*Cloning Kit for mRNA Template (Code 6143A)¿¡ Æ÷ÇÔµÈ IVT¿ë ÁÖÇü plasmid´Â BspQ I Á¦ÇÑÈ¿¼ÒÀÇ Àνļ­¿­ÀÌ Æ÷ÇԵǾî ÀÖÁö ¾Ê¾Æ »ç¿ëÀÌ ºÒ°¡´ÉÇÏ´Ù. ÀÌ Á¦Ç°À» »ç¿ëÇÒ °æ¿ì¿¡´Â Hind III (Code 1060A)¸¦ ÃßõÇÑ´Ù.

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