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Poly(A) ¹× ¹Ýº¹ ¼­¿­À» Æ÷ÇÔÇÏ´Â À¯ÀüÀÚÀÇ Å¬·Î´×¿¡ ÃÖÀû

Takara Stable Competent Cells

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Takara
9132
Takara Stable Competent Cells
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç° 
1 Set (100 §¡¡¿10)
218,400¿ø 
273,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
9132_ds_j.pdf

Á¦Ç°¼³¸í
º» Á¦Ç°Àº DNA ¾ÈÁ¤¼ºÀÌ Çâ»óµÇµµ·Ï homologous recombination°ú °ü·ÃµÈ À¯ÀüÀÚÀÇ ÀϺΰ¡ °á¼ÕµÇ¾î ÀÖ¾î poly(A) ¼­¿­À̳ª ¹Ýº¹ ¼­¿­À» °¡Áø ºÒ¾ÈÁ¤ÇÑ DNA¸¦ Ŭ·Î´×ÇÒ ¶§ ÀûÇÕÇÏ´Ù. µû¶ó¼­ Takara IVTpro¢â ½Ã¸®Áî(Code 6141 ¿Ü) µîÀÇ IVT ½ÇÇè¿¡ ¸Å¿ì È¿°úÀûÀ¸·Î »ç¿ëÇÒ ¼ö ÀÖ´Ù. ¶ÇÇÑ, ÀÌ Á¦Ç°Àº T1 bacteriophage °¨¿°¿¡ ÇÊ¿äÇÑ fhuA À¯ÀüÀÚ°¡ °á¼ÕµÇ¾î ÀÖ¾î ÇØ´ç phage¿¡ ³»¼ºÀÌ ÀÖ´Ù.

Ŭ·Î´× ½Ã, In-Fusion¢ç Snap Assembly Master Mix (Code 638947 ¿Ü)ÀÇ »ç¿ëÀ» ±ÇÀåÇϸç, pUC°è º¤ÅÍ·Î ÇüÁúÀüȯÇϸé blue/white colony screeningÀÌ °¡´ÉÇÏ´Ù.
ploy(A) ¾ÈÁ¤¼º, ¹Ýº¹ ¼­¿­ÀÇ ¾ÈÁ¤¼º ºñ±³
Competent Cells
Poly(A) length [bp]
Poly(A) full length ratio
E. coli JM109
Competent Cells
105
105
43
66
105
44
105
22
27
105
105
105
105
105
105
105
105
105
105
32
105
105
105
105
105
105
105
105
105
105
105
48
105
105
105
105
105
105
105
105
105
105
105
105
46
105
45
105
81.25£¥
(39/48)
Takara Stable
Competent Cells
105
105
105
65
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
22
105
105
105
105
105
105
105
105
105
105
105
105
105
105
105
66
105
105
105
105
105
93.75£¥
(45/48)


±×¸² 1. Poly(A) ¾ÈÁ¤¼ºÀÇ ºñ±³

105 bpÀÇ poly(A)¸¦ ÄÚµùÇÏ´Â Çö󽺹̵å DNA¸¦ E.coli JM109 Competent Cells (Code 9052)¿Í º» Á¦Ç°(Code 9132)¿¡ ÇüÁúÀüȯÇÏ°í LB Ç÷¹ÀÌÆ®¿¡¼­ ¹è¾çÇÑ ÈÄ, 48°³ÀÇ ÄݷδϿ¡¼­ Çö󽺹̵带 Á¤Á¦ÇÏ¿´´Ù. Á¤Á¦ÇÑ Çö󽺹̵带 ½ÃÄö½ÌÇÏ¿© (sanger¹ý) poly(A)ÀÇ ±æÀ̸¦ ÃøÁ¤ÇÏ¿´´Ù. Poly(A) Å»¶ôÀ¸·Î ÀÎÇØ 105 bpº¸´Ù ±æÀÌ°¡ ª¾ÆÁø poly(A)´Â »¡°£»öÀ¸·Î Ç¥½ÃÇÏ¿´´Ù.
48°³ Çö󽺹̵å Áß poly(A) Àüü ±æÀ̸¦ °®´Â Çö󽺹̵尡 JM109¿¡¼­´Â 39°³ (poly(A) full length ºñÀ² : 39/48=81.25%), Takara Stable Competent Cells¿¡¼­´Â 45°³ (poly(A) full length ºñÀ² : 45/48=93.75%)À̾ú´Ù. ÀÌ °á°ú´Â Takara Stable Competent CellsÀÌ JM109º¸´Ù poly(A)¸¦ Æ÷ÇÔÇÏ´Â À¯ÀüÀÚ Å¬·Î´×¿¡ ¾ÈÁ¤ÀûÀ̶ó´Â °ÍÀ» º¸¿©ÁØ´Ù.


±×¸² 2. ¹Ýº¹ ¼­¿­ÀÇ ¾ÈÁ¤¼º ºñ±³
Á¦ÇÑÈ¿¼Ò 󸮷ΠÀý´Ü ¹× ÃßÃâÇÒ ¼ö ÀÖ´Â 32 bp ¡¿ 15ȸ¿Í 32 bp ¡¿ 20ȸ ¹Ýº¹ ¼­¿­À» ÄÚµùÇÏ´Â Çö󽺹̵å DNA (¿ÞÂÊ ±×¸²) ¸¦ E.coli JM109 Competent Cells (Code 9052)¿Í º» Á¦Ç°(Code 9132)À¸·Î ÇüÁú ÀüȯÇÏ¿© LB Ç÷¹ÀÌÆ®¿¡ ¹è¾ç ÈÄ ¾òÀº ÄݷδϿ¡¼­ Çö󽺹̵带 Á¤Á¦ÇÏ¿´´Ù.
Çö󽺹̵åÀÇ ±æÀÌ¿Í Á¦ÇÑÈ¿¼Ò (Sal I, EcoR V, EcoR I) ó¸® ½Ã °ËÃâµÇ´Â ¹êµå¸¦ ¾Æ°¡·Î½º°Ö Àü±â¿µµ¿À¸·Î È®ÀÎÇÑ °á°ú, Á¤»óÀûÀÎ ¼­¿­À» À¯ÁöÇÏ°í ÀÖ´Â Çö󽺹̵尡 JM109´Â 96°³ cell Áß 64°³ (66.7%)ÀÎ ¹Ý¸é, Takara Stable Competent CellsÀº 120°³ Áß 97°³ (80.8%) ¿´´Ù. ÀÌ °á°ú´Â Takara Stable Competent Cells°¡ JM109º¸´Ù ¹Ýº¹ ¼­¿­À» °¡Áø À¯ÀüÀÚ¿¡ ´ëÇØ ¾ÈÁ¤¼ºÀÌ ³ô´Ù´Â °ÍÀ» º¸¿©ÁØ´Ù.
±¸¼º

Takara Stable Competent Cells

100 §¡ x 10 vials

pUC19 DNA (0.1 ng/§¡)

10 §¡

SOC Medium*

1 §¢ x 10 vials

* SOC MediumÀÇ ±¸¼º
2 %

Tryptone

0.5 %

Yeast extract

10 mM

NaCl

2.5 mM

KCl

10 mM

MgSO4

10 mM

MgCl2

20 mM

Glucose

º¸°ü
- 80¡É
Genotype
- , endA1 , supE44 , thi-1 , recA1 , relA1 , gyrA96 , phoA , ¥ÄsbcCD , ¥Õ80d lacZ¥ÄM15 , ¥ÄfhuA , ¥Ä(lacZYA - argF) U169 , ¥Ä(mrr - hsdRMS - mcrBC) , ¥ÄmcrA , ¥ë -
( K- 12 strains of E. coli)
Cell density
1¢¦2¡¿109 bacteria/§¢
ÇÃ¶ó½º¹Ìµå ½ºÅ©¸®´×¿¡ »ç¿ëµÈ Ç×»ýÁ¦ ¹× selection ³óµµ

Ç×»ýÁ¦

Ampicillin

Kanamycin

Chloramphenicol

streptomycin

tetracycline

³óµµ (§¶/§¢)

100

50

20

50

20

°ü·Ã Á¦Ç°
- Template plasmid Á¦ÀÛºÎÅÍ °í¼öÀ² IVT±îÁö Takara IVTpro¢â mRNA Synthesis System (Code 6141)
- IVT¿ë template plasmid Á¦ÀÛ kit : Cloning Kit for mRNA Template (Code 6143)
- IVT Àü vectorÀÇ ¼±ÇüÈ­¿¡ »ç¿ëÇÏ´Â Type IIS Á¦ÇÑÈ¿¼Ò: BspQ I (Code 1227A)
- In-Fusion¢ç cloning½Ã °³¼±µÈ colony Çü¼º ´É·ÂÀ¸·Î ¼º´ÉÀº up °¡°ÝÀº down In-Fusion¢ç Snap Assembly Master Mix (Code 638947)
- CloningÀ» À§ÇÑ PCR È¿¼Ò : PrimeSTAR¢ç GXL DNA Polymerase (Code R050A)
- pUC°è plasmid vector DNA Ŭ·Î´× ½Ã ½ºÅ©¸®´× : X-Gal (5-Bromo-4-Chloro-3-Indolyl-¥â- D -Galactoside (Code 9031)

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