SMART cDNA Library Construction Kit´Â phage ¥ëTriplEx2 vector¿¡ full-length cDNA¸¦ cloningÇÏ´Â Á¦Ç°ÀÌ´Ù. ÀÌ Á¦Ç°Àº cDNAÁõÆøÀ» À§ÇÑ SMART ±â¼ú°ú adaptor ¾øÀÌ ¥ëTriplEx2 vector¿¡ cloningÇÏ´Â ±â¼úÀ» ÀÌ¿ëÇϸç ÃÊ±â ½Ã·á ¾ç¿¡ µû¶ó µÎ °¡Áö ½ÇÇè °úÁ¤À¸·Î ³ª´²Á® ÀÖ´Ù. ù ¹øÂ° °úÁ¤Àº »õ·Ó°Ô °í¾ÈµÈ long-distance PCR(LD-PCR) ¹æ¹ýÀ¸·Î 50 ngÁ¤µµÀÇ ÀûÀº ¾çÀÇ RNA ½Ã·á¿¡ Àû¿ëÇÒ ¼ö ÀÖ´Ù (1). µÎ ¹øÂ° °úÁ¤Àº Á» ´õ ÀϹÝÀûÀÎ °úÁ¤À¸·Î ºñ±³Àû ¸¹Àº ¾çÀÇ Ãʱ⠽÷á (¿¹¸¦ µé¸é, 1 ¥ìg ¶Ç´Â ±× ÀÌ»óÀÇ poly A+ RNA)À» »ç¿ëÇÏ´Â ½ÇÇèÀÚ¿¡°Ô ÀûÇÕÇÏ´Ù. µÎ °úÁ¤ ¸ðµÎ ¸¹Àº ¾çÀÇ full-lengthÀÇ double-strand cDNA¸¦ ÇÕ¼ºÇϱâ À§ÇÏ¿© 1st strand ÇÕ¼º°úÁ¤¿¡¼­ SMART IV Oligonucleotide¸¦ ÀÌ¿ëÇÑ´Ù. µÎ ½ÇÇè °úÁ¤ ¸ðµÎ UserManual¿¡ Ç¥±âµÇ¾î ÀÖÀ¸¸ç °¢ÀÚÀÇ ½ÇÇè¿¡ ¸Â°Ô ¼±ÅÃÇϵµ·Ï ÇÑ´Ù. °¢°¢ÀÇ kit´Â cDNA library 7ȸ Á¦ÀÛ ºÐÀÌ´Ù.
¶ÇÇÑ, Æ÷À¯·ù ¹ßÇö¿ë vectorÀÎ pEXP-Lib, retroviral expression vectorÀÎ pRetro-Lib µî SMART cDNA Library Construction Kit¿Í ÇÔ²² »ç¿ëÇÒ ¼ö ÀÖ´Â 7°¡Áö vector°¡ º°µµ ÆÇ¸ÅµÇ°í ÀÖ´Ù.

SMART library´Â ±âÁ¸ÀÇ ÀüÅëÀûÀÎ ¹æ¹ý¿¡ ºñÇØ ¸Å¿ì ³ôÀº È¿À²·Î Àüü±æÀ̸¦ Æ÷ÇÔÇÏ´Â cloneÀ» Á¦ÀÛÇÒ ¼ö ÀÖ´Ù. µû¶ó¼­ SMART cDNA library¿¡¼­ ¼±º°µÈ cloneÀº mRNAÀÇ 5¡¢-untranslated regionÀ» ¿ÏÀüÈ÷ Æ÷ÇÔÇϰԵȴ٠(2).

RNA/cDNA Quality Assay¸¦ »ç¿ëÇÏ¸é ½ÇÇèÀ» ½ÃÀÛÇϱâ Àü¿¡ Ãʱ⠽÷á·Î »ç¿ëµÇ´Â human RNAÀÇ »óŸ¦ È®ÀÎÇÒ ¼ö ÀÖ´Ù. º» ½ÇÇè¿¡´Â RT-PCR¹ýÀÌ ÀÌ¿ëµÇ¹Ç·Î ½ÇÇè¿¡ Àû¿ëÇÒ full-length cDNA°¡ ÁõÆøµÇ´ÂÁö ¿©ºÎ¸¦ Á÷Á¢ÀûÀ¸·Î È®ÀÎÇÒ ¼ö ÀÖ´Ù. ±âÁ¸ÀÇ ½ÇÇè½Ç¿¡¼­ »ç¿ëÇÏ´ø ÀåºñµéÀ» ÀÌ¿ëÇϹǷΠºü¸£°Ô RNA »óŸ¦ È®ÀÎÇÒ ¼ö ÀÖÀ» »Ó¸¸ ¾Æ´Ï¶ó ºÒÆíÇϰí À¯ÇØÇÑ formaldehyde gelÀ» Á¦ÀÛÇÒ Çʿ䵵 ¾ø´Ù.


Figure 1. Comparison of SMART cDNA synthesis vs. conventional synthesis for library construction.

Á¦Ç°¸í Cat. No. ¿ë·® ¸Å´º¾ó First Strand Synthesis cDNA Amplification Vector Ư¡
SMART cDNA
Library Construction
Kit
634901 10 ȸ ¸Å´º¾ó SMART IV Oligonucleotide 5' PCR Primer ¥ëTriplEx2
(lamda phage)
* SMART cDNA ÇÕ¼º ±â¼ú
* lamda phage¿¡ library Á¦ÀÛ
* Á¦ÀÛµÈ library¸¦ °í°´ÀÌ ¿øÇÏ´Â plasmid·Î coversion±ÇÀåÇÏÁö ¾ÊÀ½
CDS III/3' PCR Primer
In-Fusion SMARTer
Directional
cDNA Library
Construction Kit
634933 10 ȸ ¸Å´º¾ó SMARTer V Oligonucleotide 5' PCR Primer II A pSMART2IFD
Linearized
Vector
* SMART񃬣 In-Fusion Cloning 񃬣
* ¿øÇÏ´Â vector¿¡ °ð¹Ù·Î library
Á¦ÀÛ °¡´É(È¿À² °í·Á ÇÊ¿ä)
* InsertÀÇ cloning ¹æÇ⼺ êó
3¡¯ In-Fusion SMARTer CDS Primer 3¡¯ In-Fusion SMARTer PCR Primer