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º» Á¦Ç°Àº °£´ÜÇÑ ¹æ¹ýÀ¸·Î PCR »ê¹°À» ÆòÈ° ¸»´Ü (blunt end)ÀÇ º¤ÅÍ¿¡ cloning ÇÏ´Â Á¦Ç°ÀÌ´Ù. PCR »ê¹°ÀÇ ÆòÈ° ¸»´ÜÈ (blunting)¿Í 5¡¯ ¸»´Ü ÀλêÈ (5¡¯-phosphorylation)¸¦ µ¿½Ã¿¡ ÁøÇàÇϱ⠶§¹®¿¡ ÆòÈ° ¸»´ÜÀÇ PCR »ê¹°°ú dA°¡ Ãß°¡µÈ PCR »ê¹° ¸ðµÎ ÇѹøÀÇ ¹ÝÀÀÀ¸·Î ligation ÇÒ ¼ö ÀÖ´Ù. º» Á¦Ç°Àº PCR »ê¹°ÀÇ º°µµÀÇ Àüó¸® °úÁ¤ ¾øÀÌ (¿¹. È¿¼Ò ºÒÈ°¼ºÈ, ÀÜ¿© dNTP ¹× primer Á¦°Å µî) blunting kination ¹ÝÀÀ¿¡ ÀÌ¿ëÇÒ ¼ö ÀÖ´Ù. Ligation ¹ÝÀÀÀÌ ¸Å¿ì È¿À²ÀûÀÎ DNA Ligation Kit £¼Mighty Mix£¾ (Code 6023)À» »ç¿ëÇÏ°í Àֱ⠶§¹®¿¡ ºü¸£°í °£´ÜÇÑ ligationÀÌ °¡´ÉÇÏ´Ù. ÀÌ Á¦Ç°Àº PrimeSTAR¢ç GXL DNA Polymerase (Code R050A)¿Í °°Àº È¿¼Ò·Î ÁõÆøµÈ Blunt endÀÇ PCR »ê¹°ÀÇ cloning¿¡ »ç¿ëÇÒ ¼ö ÀÖ´Ù.
³»¿ë (20ȸ¿ë)
10 X Blunting Kination Buffer |
40 §¡ |
Blunting Kination Enzyme Mix |
20 §¡ |
Ligation Mighty Mix *1 |
120 §¡ |
Control Vector (pUC118 Hinc II /BAP) (50 ng/§¡) |
20 §¡ |
Control Insert *2 (200 ng/§¡) |
10 §¡ |
ddH2O |
340 §¡ |
*1 DNA Ligation Kit £¼Mighty Mix£¾(Code 6023)¿¡ Æ÷ÇÔµÈ ½Ã¾à°ú µ¿ÀÏ
*2 ¥ëDNAÀ» ÁÖÇüÀ¸·Î
TaKaRa Ex Taq¢ç¿¡¼ ÁõÆøµÈ 500 bpÀÇ PCR »ê¹°
º¸Á¸ -20¡É
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- PCR »ê¹°À» ÆòÈ° ¸»´Ü (blunt end)ÀÇ vector¿¡ cloning
pUC118 Hinc II / BAP
pUC118 Hinc II / BAP´Â Hinc IIIÀ» ÀÌ¿ëÇÏ¿© pUC118 DNA¸¦ Àý´Ü ÈÄ E. coli C75¿¡¼ Á¤Á¦µÈ alkaline phosphatase (BAP)¿¡ ÀÇÇØ Å»ÀλêÈÇÑ ÆòÈ° ¸»´Ü (blunt end) cloning vectorÀÌ´Ù.
- Vector map of pUC118
- Cloning site of pUC118
°ü·ÃÁ¦Ç°
- ´Ù¾çÇÑ »ùÇÿ¡ Àû¿ë °¡´ÉÇÑ 1st choice PCR È¿¼Ò TaKaRa Ex Premier DNA Polymerase (Code RR370A)
- Long fragment ÁõÆø ¹× GC-rich template ÁõÆø¿¡ °ÇÑ High Fidelity PCR È¿¼Ò PrimeSTAR¢ç GXL DNA Polymerase (Code R050A)
- ÃÖ°í ·¹º§ÀÇ Á¤È®¼º £¦ ÃÖ°í ¼ÓµµÀÇ ½ÅÀ强 PrimeSTAR¢ç Max DNA Polymerase (Code R045A)
Âü°íÀÚ·á
- Cloning ½ÇÇè ÀÔ¹®ÀÚ¸¦ À§ÇÑ ¡®Step by Step Cloning °¡À̵塯
- ½ÇÇè ¸ñÀûº°·Î ÃßõÇÏ´Â Cloning Kit