miRNA Cloning3' Cloning Linkers»õ·Î¿î miRNA¸¦ isolationÇÏ´Â °¡Àå Á÷Á¢ÀûÀÎ ¹æ¹ýÀº Ŭ·Î´×¿¡ ÀÇÇÑ °ÍÀÔ´Ï´Ù. ÀÌ·¯ÇÑ ¹æ¹ýÀº cloning linkers¸¦ miRNA¿¡ ligationÇÏ´Â ÀÛ¾÷ÀÌ
ÇÊ¿äÇÕ´Ï´Ù. Adenlyation¿¡ ÀÇÇؼ pre-activated µÈ ¸µÄ¿¸¦ »ç¿ëÇϸé Çâ»óµÈ cloning efficiency¸¦ ¾òÀ» ¼ö ÀÖ½À´Ï´Ù. IDT´Â BanI cloning site¸¦ Æ÷ÇÔÇϸç ddC¿¡ ÀÇÇØ 3'-end°¡ blockµÇ°í 5'-end ¿¡ adenylyl activatedµÈ Ready Made linker oligonucleotide ¸¦ Á¦°øÇÕ´Ï´Ù. ÀÌ°ÍÀº miRNA cloningÀ̳ª RNA ligase¸¦ »ç¿ëÇÏ´Â ºñ½ÁÇÑ application¿¡ ¹Ù·Î »ç¿ëµÉ ¼ö ÀÖ½À´Ï´Ù
Linker-1Àº original modban sequence·Î BanI restriction site°¡ Æ÷ÇԵǾî ÀÖ½À´Ï´Ù.
Linker-2 ÀºAvaI °ú StyI restriction sites°¡ Æ÷ÇԵǾî ÀÖ½À´Ï´Ù.
Linker-3 ÀºEcoRI °ú MspI restriction sites°¡ Æ÷ÇԵǾî ÀÖ½À´Ï´Ù.
ÀÌ ¼¼ °¡Áö linkerµéÀº self ligationÀ» ¸·±â À§ÇØ 3'-terminal dideoxy-C (ddc)base·Î modification µÇ¾î ÀÖ°í cloning¿¡ ¹Ù·Î »ç¿ëÇÒ ¼ö ÀÖµµ·Ï lyophoilizedµÇ¾î Á¦°øµË´Ï´Ù. µû¶ó¼ ATP¾øÀÌ T4 RNA Ligase¸¦ ÀÌ¿ëÇÏ¿© ligation mix¿¡ ¿øÇϽô ³óµµ·Î ¼¯¾î¼ »ç¿ëÇÏ½Ã¸é µË´Ï´Ù.
Modifications: 5'-Adenylated; fully activated and ready for use
3'-end blocked with a dideoxy-C base
Purification: HPLC purified
Quality Control: Identity confirmed by ESI Mass Spectrometry
Tested for reactivity with RNA ligase
Certificate of Analysis for each linker available online here
Custom AdenylationT4 RNA Ligase ´Â single-strand nucleic acid sequence¸¦ adenylationÇϱâ À§ÇØ ATP¸¦ »ç¿ëÇÕ´Ï´Ù. ÀÌ·¸°Ô activated µÈ adenylated-oligo´Â °øÀ¯°áÇÕÀ» ÅëÇØ µÎ¹ø° single-stranded sequence 3'-OH ¿¡ ¿¬°áµË´Ï´Ù. Pyrophosphate linkage¸¦ Æ÷ÇÔÇÑ adenylated oligonucleotides´Â ATP°¡ ¾ø´Â »óȲ¿¡¼µµ T4 RNA LigaseÀÇ substrates°¡ µË´Ï´Ù. IDT´Â chemical adenylation method¸¦ »ç¿ëÇÏ¿© ¿Ã¸®°í¿¡ custom adenylationÀ» Á¦°øÇÏ¿© RNA-Ligase¿¡ »ç¿ëÇÒ ¼ö ÀÖµµ·Ï ÇÕ´Ï´Ù. T4 RNA Ligase´Â adenylated DNA linker ¸¦ adenylated RNA linker¿Í °°Àº È¿À²·Î »ç¿ëÇϱ⠶§¹®¿¡ IDT¿¡¼´Â adenylated DNA oligos ¸¦ ÃßõÇÕ´Ï´Ù. ¶ÇÇÑ IDT ¿¡¼´Â circularizationÀ» ¹æÁöÇϱâ À§ÇÏ¿© adenylated oligoÀÇ 3'-end¿¡ blockingÀÌ ÇÊ¿äÇÕ´Ï´Ù. BlockingÀ» À§Çؼ´Â 3'-Spacer C3 /3SpC3/ or dideoxycytosine /3ddC/ µîÀÌ »ç¿ëµË´Ï´Ù.
5' M.R.S (Multiple Restriction Site) miRNA Cloning Linker5' M.R.S Linker sequence´Â ¾î¶² 3'miRNA Cloning linker¿Íµµ »ç¿ëÇÒ ¼ö ÀÖµµ·Ï µðÀÚÀÎ µÇ¾ú½À´Ï´Ù. ½ÃÄö½º´Â 5' phosphate group¸¦ Æ÷ÇÔ
ÇÏ´Â RNAÀÇ 5' end¿¡ linking µÇµµ·Ï ÃÖÀûÈ µÇ¾ú½À´Ï´Ù. ¹ÝÀÀÀº 1mM ATP ÇÏ¿¡¼ T4 RNA Ligase¿¡ ÀÇÇؼ ÀÌ·ç¾îÁý´Ï´Ù. ½ÃÄö½º¿¡´Â Á¦ÇÑ
È¿¼Ò »çÀÌÆ®°¡ ÀÖ¾î Ban1 (Linker 1), StyI and AvaI (Linker 2) and EcoRI (Linker 3)°ú °°ÀÌ »ç¿ëÇÒ ¼ö ÀÖ½À´Ï´Ù. Doubly linked RNA°¡ reverse transcription µÇ¸é 3' cloning linker ¿¡ ÇØ´çÇÏ´Â Á¦ÇÑÈ¿¼Ò°¡ 5' M.R.S Linker sequence ¿¡ ȣȯÀûÀÎ ¸»´ÜÀ» ¸¸µé¾î³»¾î concatamerization°ú Ŭ·Î´×ÀÌ ÀϾ°Ô µË´Ï´Ù. ´õ¿íÀÌ M.R.S Linker ¿¡ ÀÖ´Â Ãß°¡ÀûÀÎ Á¦ÇÑÈ¿¼Ò »çÀÌÆ®°¡ specific 3' linkers¿Í ¸ÅÄ¡µÉ °æ¿ì ends for directional cloning À» À§ÇÑ ¸»´ÜÀ» ¸¸µé¾î ³¾ ¼ö ÀÖ½À´Ï´Ù. ¿¹¸¦ µé¾î EcoRI °ú BanIÀ¸·Î M.R.S Linker/Linker 3 digestion Çϸé BanI 5' end ¿Í EcoRI 3' ¸¦ ³²±â°Ô µË´Ï´Ù.
miSPIKE 21-mer Internal RNA ControlmiSPIKE Àº synthetic 21-mer RNA·Î GenBank, miRBase, ¶Ç´Â RNAdb¿¡ ÀÖ´Â ¾î¶² ¾Ë·ÁÁø RNA sequence¿Íµµ match°¡ µÇÁö ¾Ê´Â ½ÃÄö½º
¸¦ °¡Áý´Ï´Ù. ÀÌ RNA oligonucleotide´Â 5' phosphate°¡ ¾øÀÌ ÇÕ¼ºÀÌ µÇ¾î denaturing acrylamide gel ¿¡¼ small RNA enrichment ¸¦ À§ÇÑ size marker¿Í 3' ligation control ·Î »ç¿ëµÉ ¼ö ÀÖÀ¸¸ç 5' ligationÀº µÉ ¼ö ¾ø½À´Ï´Ù.
Sequence: 5'- rCrUrCrArGrGrArUrGrGrCrGrGrArGrCrGrGrUrCrU -3'
piSPIKE 31-mer Internal RNA ControlpiSPIKE Àº synthetic 31-mer RNA·Î miSPIKE¿¡¼ 10base ´õ ±æ°í ¿ª½Ã 5' phosphate°¡ ¾ø½À´Ï´Ù.
Sequence: 5'- rCrUrCrArGrGrArUrGrGrCrGrGrArGrCrGrGrUrCrUrCrArCrUrGrArArCrGrU -3'
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