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Cycloner, Benchtop Equivalent Simulated Cloning Process![]() CloningWizard ¹Ù·Î °¡±â Cycloner´Â ½ÇÁ¦ ½ÇÇèÇϽô ºÐµéÀ» À§ÇØ ¸¸µé¾îÁø Ŭ·Î´× ½Ã¹Ä·¹ÀÌ¼Ç ÆÐŰÁöÀÔ´Ï´Ù. Cycloner¸¦ ÀÌ¿ëÇÏ¿© Ŭ·Î´×À» ÇϱâÀüÀ̳ª ½ÇÇè Áß¿¡ ÀϾ ¼ö ÀÖ´Â ºÒÇÊ¿äÇÑ ÀÛ¾÷À̳ª ½ÇÇè ¿¡·¯µéÀ» ÁÙÀÏ ¼ö ÀÖ½À´Ï´Ù. Cycloner´Â PrimerDesigner*, PCRcycler*, Cutter*, A-Ligator*, Cut&Ligation*, Recombination*, HRecombination*, Blunt-It*, SeqEditor*ÀÇ µ¶¸³ÀûÀÎ ÇÁ·Î±×·¥ Åø·Î ±¸¼ºµÇ¾îÀÖ½À´Ï´Ù. À̵é ÇÁ·Î±×·¥µéÀº PCR primer¸¦ ¼³°èÇϰųª Á¦ÇÑÈ¿¼Ò À§Ä¡¸¦ ºÐ¼®ÇÏ´Â °³º°ÀÛ¾÷¿¡µµ È¿°úÀûÀ¸·Î »ç¿ëµÉ ¼ö ÀÖÀ¸³ª, ¼·Î ¿¬°èÇÏ¿© ½ÇÇè½Ç¿¡¼ ÀÌ·ç¾îÁö´Â ÀüüÀûÀΠŬ·Î´× ÀÛ¾÷À» ½Ã¹Ä·¹ÀÌ¼Ç Çϴµ¥ ¸Å¿ì À¯¿ëÇÏ°Ô »ç¿ëÇÒ ¼ö ÀÖ´Â ´º·ÎÁ¦³Ø½ºÀÇ µ¶Ã¢ÀûÀÎ ¼ÒÇÁÆ®¿þ¾îÀÔ´Ï´Ù. ¿Ïº®ÇÑ Å¬·Î´× ¼Ö·ç¼ÇÀÎ Cycloner¸¦ º¸´Ù È¿À²ÀûÀ¸·Î »ç¿ëÇϽ÷Á¸é, À¯Àú°¡À̵忡 ¼³¸íµÈ Neurogenex Sequence Format (NGSF), Cycloner TutorialÀ» Àо½Ê½Ã¿À. PrimerDesigner*´Â PCR primer¸¦ µðÀÚÀÎÇϱâ À§ÇØ °³¹ßµÈ ÇÁ·Î±×·¥ ÅøÀÔ´Ï´Ù. PrimerDesigner*´Â Primer¸¦ µðÀÚÀÎÇϰųª PCR°úÁ¤ÀÇ ³ë·Â°ú ½Ç¼ö¸¦ ÃÖ¼ÒÈÇϵµ·Ï ¸¸µé¾îÁ³½À´Ï´Ù. ¿¹¸¦ µé¾î, PrimerDesigner*´Â primer¿¡ enzyme linkerÀÇ Ãß°¡¿Í ³»ºÎ enzyme siteÀÇ ÀÚµ¿Ã¼Å© µî ¸¹Àº ºÎºÐÀ» Áö¿øÇÕ´Ï´Ù. PCRcycler*´Â ÀÌ ÈÄ Å¬·Î´× °úÁ¤À» ÁøÇàÇϱ⠻ç¿ëµÇ´Â ½ÃÄö½º¸¦ ¸¸µé¾î³À´Ï´Ù. °£´ÜÈ÷ copy&paste¸¦ ÇÏ¿© ÁõÆøµÈ PCR product ½ÃÄö½º¸¦ ¸¸µé ¼ö ÀÖ½À´Ï´Ù. Cutter*¿Í A-Ligator*´Â ÀϹÝÀûÀÎ DNA Ŭ·Î´× °úÁ¤À» ½Ã¹Ä·¹À̼ÇÇÕ´Ï´Ù. ½ÇÇè½Ç¿¡¼ enzyme digestion, gel running, elutionÀ» ÅëÇØ º¤ÅÍ¿Í insert DNA¸¦ ÁغñÇÕ´Ï´Ù. ÀÌ·¯ÇÑ Å¬·Î´×À» ÇϰíÀÚ ÇÏ´Â ½ÃÄö½º¿Í ÀûÀýÇÑ Á¦ÇÑÈ¿¼Ò¸¦ ÀÔ·ÂÇÔÀ¸·Î½á °£´ÜÇÏ°Ô ½Ã¹Ä·¹ÀÌ¼Ç ÇÒ ¼ö ÀÖ½À´Ï´Ù. A-Ligator*´Â º¤ÅÍÀÇ enzyme site(overhang, blunt, T addition µîÀ» Æ÷ÇÔ)¿Í insert ½ÃÄö½º¸¦ ÀÚµ¿À¸·Î ÀνÄÇÏ¿©, Ŭ·Î´×µÈ ½ÃÄö½º¸¦ »ý¼ºÇÕ´Ï´Ù. ÀÌ ½ÃÄö½º¸¦ SeqSenser ÆÐŰÁö¿¡ ÀÖ´Â RestrictionAnalyzer³ª EMBOSS ÆÐŰÁöÀÇ alignment ¼ÒÇÁÆ®¿þ¾îÀÎ water³ª Blast °Ë»öÀ» ÀÌ¿ëÇÔÀ¸·Î½á ´õ ÀÚ¼¼È÷ ºÐ¼®ÇÒ ¼ö ÀÖ½À´Ï´Ù. Cut&Ligation´Â ½ÃÄö½º¸¦ ÁÖ¾îÁø enzymeÀ¸·Î Àß¶ó »ý±â´Â FragmentµéÀ» ±æÀ̸¦ ±âÁØÀ¸·Î ¼±ÅÃÇÏ¿© ligation¹ÝÀÀÀ» ½Ãŵ´Ï´Ù. CloningWizard´Â Vector Cut , PCR, Insert Cut, Ligationµî Ŭ·Î´× ÀÛ¾÷ÀÇ ½ÃÀÛºÎÅÍ Ligation Product¸¦ ¾ò´Â °úÁ¤ÀÇ Àü¹ÝÀûÀÎ ¿¹ÃøÀ» °¡´ÉÇÏ°Ô ÇÕ´Ï´Ù. RecombinationÀº ¶÷´Ù ÆÄÁö À¯·¡ÀÇ ÀçÁ¶ÇÕ ½ÃÄö½º°£ÀÇ À§Ä¡ ƯÀÌÀû ÀçÁ¶ÇÕ ¹ÝÀÀÀ» ½Ã¹Ä·¹À̼ÇÇÏ¿© ¹ÝÀÀ °á°ú ½ÃÄö½º¸¦ ¸¸µé¾î ÁÝ´Ï´Ù. HRecombinationÀº µÎ°³ÀÇ DNA ½ÃÄö½ºÀÇ Homologous Recombination °á°ú¸¦ ±¸ÇÕ´Ï´Ù. Blunt-ItÀº ½ÃÄö½ºÀÇ ³¡ºÎºÐÀ» ¹¶ÅöÇÏ°Ô ÇÕ´Ï´Ù Cycloner´Â ´ç½ÅÀÇ ¿¬±¸¸¦ ÇÑÃþ ´õ µµ¿ÍÁÙ °ÍÀÔ´Ï´Ù. |
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