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TaKaRa LA Taq¢çÀº LA PCR ¿ø¸®¸¦ ÀÀ¿ëÇÑ 3¡¯¡æ 5¡¯exonuclease Ȱ¼º (proof reading Ȱ¼º)À» °®´Â ³»¿­¼º DNA polymeraseÀÌ´Ù. ÀϹÝÀûÀÎ PCR Á¶°Ç¿¡¼­ ±âÁ¸ÀÇ Taq polymerase¿¡ ºñÇÏ¿© ³ôÀº ÁõÆøÈ¿À²À» º¸À̸ç, ±âº»ÀûÀ¸·Î ¾î¶°ÇÑ ¹ÝÀÀ¿¡µµ Àû¿ëÇÒ ¼ö ÀÖÀ¸³ª ƯÈ÷ 15 kb ÀÌ»óÀÇ ÁõÆø¿¡ È¿°úÀûÀÌ´Ù.
º» Á¦Ç°Àº TaKaRa LA Taq¢ç¿¡ Anti-Taq antibody¸¦ ÀÌ¿ëÇÑ Hot Start PCR¿ë È¿¼ÒÀ̸ç, °í¿Â¿¡ µµ´ÞÇÒ ¶§±îÁö´Â antibody°¡ °áÇÕÇϰí ÀÖ¾î polymerase Ȱ¼ºÀ» ¾ïÁ¦ÇÑ´Ù. µû¶ó¼­ PCR cycleÀ» ½ÃÀÛÇϱâ Àü¿¡ ¹ß»ýÇÒ ¼ö ÀÖ´Â misprimingÀ̳ª primer dimer¿¡ ÀÇÇÑ ºñƯÀÌÀû ÁõÆøÀ» ¹æÁöÇÒ ¼ö ÀÖ´Ù.
Anti-Taq antibody´Â PCR cycleÀÇ ÃÖÃÊ DNA denaturation ´Ü°è¿¡¼­ º¯¼ºµÇ¹Ç·Î Anti-Taq antibodyÁ¦°Å¸¦ À§ÇÑ Ãß°¡ °úÁ¤Àº ÇÊ¿äÇÏÁö ¾Ê°í, ±âÁ¸¿¡ »ç¿ëÇÏ´ø PCR Á¶°ÇÀ» ±×´ë·Î »ç¿ëÇÑ´Ù.
³»¿ë (250 U)
1. TaKaRa LA Taq HS (5 U/§¡)
25 §¡
2. 10¡¿ LA PCR Buffer II (Mg2+ plus, 25 mM)
1 §¢
3. dNTP Mixture (°¢ 2.5 mM)
400 §¡
º¸Á¸ -20¡É
Çü»ó

20 mM

Tris-HCl (pH8.0)

100 mM

KCl

0.1 mM

EDTA

1 mM

DTT

0.5%

Tween 20

0.5%

NP-40

50%

Glycerol

÷ºÎ dNTP Mixture Á¶¼º*

³óµµ

°¢ 2.5 mM

Çü»ó

¼ö¿ë¾× (³ªÆ®·ý¿°), pH7~9

¼øµµ

°¢ 98% ÀÌ»ó

*dATP, dCTP, dGTP, dTTPµî ¸ô È¥ÇÕ¹°·Î Èñ¼®ÇÏÁö ¾Ê°í ±×´ë·Î PCR¹ÝÀÀ¿¡ ÀÌ¿ëÇÒ ¼ö ÀÖ´Ù.
PCR »ê¹°¿¡ ´ëÇØ
TaKaRa Ex Taq¢ç HS¸¦ ÀÌ¿ëÇÏ¿© ÁõÆøÇÑ PCR »ê¹°ÀÇ ´ëºÎºÐÀº 3¡¯ ¸»´Ü¿¡ A°¡ 1¿°±â ºÎ°¡µÇ¾î ÀÖ´Ù. µû¶ó¼­ ÀÌ PCR »ê¹°À» ±×´ë·Î T-vector pMD20 (Code 3270) ¶Ç´Â T-vector pMD19 (Simple) (Code 3271)¿¡ cloningÇÒ ¼ö ÀÖ´Ù. ¶ÇÇÑ ¸»´Ü ÆòȰȭ ¹× ÀλêÈ­ ÈÄ, ÆòȰ ¸»´Ü vector¿¡ cloningÇÒ ¼öµµ ÀÖ´Ù.
°ü·ÃÁ¦Ç°
- GC rich ¼­¿­ÀÇ ÁõÆø¿¡ Ź¿ù: TaKaRa LA Taq¢ç with GC Buffer
- 2x PCR master mix: One Shot LA PCR¢â Mix Ver. 2.0
- ¿ªÀü»ç¹ÝÀÀÀ» À§ÇÑ all-in-One kit: PrimeScript¢â 1st strand cDNA Synthesis Kit
- 5x premix ¿ªÀü»ç ½Ã¾à: PrimeScript¢â IV 1st strand cDNA Synthesis Mix
- ´Ù¾çÇÑ »ùÇÃÀÇ DNA Á¤Á¦¿¡ ÃÖÀû: TaKaRa MiniBEST Universal Genomic DNA Extraction Kit
- ¿Ã¶ó¿îµå Agarose: SeaKem¢ç LE Agarose
- 5ºÐ¸¸¿¡ Àü±â¿µµ¿ ¿Ï·á! Precast gel system: FlashGel¢â System
- ÄÄÆÑÆ®ÇÑ 96 well gradient PCR ±â±â : Clontech PCR Thermal Cycler GP