[In-Fusion Cloning¿¡ °üÇÏ¿©]
Q1. In-Fusion CloningÀÌ ¹«¾ùÀΰ¡¿ä?
In-Fusion CloningÀº insertÀÇ ¾ç ¸»´Ü°ú ¼±ÇüÈµÈ º¤ÅÍ (linearized cloning vector)ÀÇ ¾ç ¸»´ÜÀ» À¶ÇÕ½ÃÄÑ ¿¬°á½ÃŰ´Â ¸Å¿ì È¿À²ÀûÀΠŬ·Î´× ¹æ¹ýÀÔ´Ï´Ù (Ligation-independent cloning method).
Q2. In-Fusion CloningÀÇ È¿À²Àº ¾î¶»°Ô µÇ³ª¿ä?
ÇϳªÀÇ insert¸¦ ÇϳªÀÇ º¤ÅÍ¿¡ Ŭ·Î´×ÇÏ´Â °æ¿ì, ÃÖ¼Ò 95% ÀÌ»óÀÇ Å¬·Î´× È¿À²À» º¸ÀÔ´Ï´Ù. Ŭ·Î´× È¿À²Àº ´Ü¼øÈ÷ ÄݷδÏÀÇ °³¼ö¸¦ ÃøÁ¤ÇÏ´Â
ÇüÁúÀüȯȿÀ² (transformation efficiency)°ú´Â ´Þ¸®, ¹ÝÀÀ ÈÄ¿¡ ¾ò¾îÁø Ŭ·ÐÀÌ insert¸¦ ¾ó¸¶³ª Á¤È®(accurate)ÇÏ°Ô Æ÷ÇÔÇϰí ÀÖ´ÂÁö¸¦ ÀǹÌÇÏ´Â ¼öÄ¡ÀÔ´Ï´Ù.
Q3. In-Fusion CloningÀº ¾î¶² ¿ø¸®·Î Ŭ·Î´× Çϳª¿ä?
In-Fusion Cloning ¹ÝÀÀÀ» À§Çؼ´Â cloning insert¿Í linearized vectorÀÇ ¾ç ¸»´Ü 15 bp¸¦ µ¿ÀÏÇÏ°Ô ¸ÂÃß¾îÁÖ´Â ÀÛ¾÷ÀÌ ÇÊ¿äÇÕ´Ï´Ù. 15 bp »óµ¿¼¿Àº
PCR ÁõÆøÀ» ÅëÇÏ¿© insertÀÇ ¾ç ¸»´Ü¿¡ ºÎ°¡ÇÒ ¼ö ÀÖ½À´Ï´Ù. In-Fusion Enzyme mix´Â cloning insert¿Í linearized vectorÀÇ ¸»´Ü 15 bp ¼¿À» 15bp ±æÀÌÀÇ
single stranded 5¡¯-overhangÀ¸·Î ¸¸µé¸ç, ÀÌ·¸°Ô »ý¼ºµÈ overhang ¼¿ÀÌ ¼·Î »óº¸ÀûÀ¸·Î °áÇÕÇÏ¿© recombinant circular construct¸¦ Á¦ÀÛÇÕ´Ï´Ù.
Recombinant construct¿¡ »ý¼ºµÈ nickÀº
E.coli competent cell ³»¿¡¼ ¼öº¹(repair)µË´Ï´Ù.
±×¸². Overview of In-Fusion Cloning Protocol
Q4. In-Fusion Cloning Á¦Ç°ÀÇ Á¾·ù°¡ ¾ÆÁÖ ´Ù¾çÇѵ¥, ¾î¶»°Ô ¼±ÅÃÇØ¾ß Çϳª¿ä?
In-Fusion CloningÀº Á¦Ç° ³» Æ÷ÇÔµÈ ±¸¼ºÇ°ÀÇ Á¾·ù¿¡ µû¶ó ¾Æ·¡¿Í °°ÀÌ ºÐ·ùµÇ¾îÀÖ°í, In-Fusion Cloning¿¡ ÃÖÀûÈµÈ competent cell, Á¤Á¦Å°Æ®, high-fidelity PCR È¿¼Ò¸¦
ÇÔ²² »ç¿ëÇÏ½Ã¸é ´õ¿í ³ôÀº È¿À²À» ±â´ëÇÒ ¼ö ÀÖ½À´Ï´Ù.
[Liquid Type Á¦Ç°Á¾·ù]
¸ðµç Á¦Ç° ³»¿¡´Â 5X In-Fusion HD Enzyme Premix, linearized pUC19 Control Vector (50 ng/§¡), 2 kb Control Insert (40 ng/§¡)°¡ Æ÷ÇԵǾîÀÖ½À´Ï´Ù.
[µ¿°á°ÇÁ¶ Á¦Ç°Á¾·ù]
¸ðµç Á¦Ç° ³»¿¡´Â µ¿°á°ÇÁ¶ ŸÀÔÀÇ È¿¼Ò In-Fusion EcoDry Mix, linearized pUC19 Control Vector (50 ng/§¡), 2 kb Control Insert (40 ng/§¡)°¡ Æ÷ÇԵǾîÀÖ½À´Ï´Ù.
Q5. In-Fusion HD Cloning kit¿Í In-Fusion HD EcoDry Cloning kitÀÇ Â÷ÀÌÁ¡Àº ¹«¾ùÀΰ¡¿ä?
In-Fusion HD Cloning Kit´Â liquid ÇüÅÂÀÇ In-Fusion HD Enzyme Premix¸¦ Æ÷ÇÔÇϰí ÀÖ´Â ¹Ý¸é, In-Fusion EcoDry Cloning Kit´Â µ¿°á °ÇÁ¶µÈ In-Fusion HD Enzyme Premix°¡
8-strip tube¿¡ ºÐÁÖµÈ ÇüÅ·ΠÁ¦°øµË´Ï´Ù. EcoDry´Â ½Ç¿Â¿¡¼ º¸°ü °¡´ÉÇÒ »Ó¸¸ ¾Æ´Ï¶ó, ¹Ì¸® ºÐÁֵǾîÀÖÀ¸¹Ç·Î handling error¸¦ ÃÖ¼ÒÈÇÒ ¼ö ÀÖ½À´Ï´Ù. Liquid ÇüÅÂÀÇ °æ¿ì 15ºÐ,
±×¸®°í EcoDry ÇüÅÂÀÇ °æ¿ì¿¡´Â 30ºÐÀÇ ¹ÝÀÀ ½Ã°£ÀÌ ÇÊ¿äÇÕ´Ï´Ù.
Q6. Cloning Enhancer (CE)°¡ ¹«¾ùÀΰ¡¿ä?
Cloning Enhancer (CE)´Â PCR ¹ÝÀÀ ÈÄÀÇ ÀÜ¿©¹°À» Á¦°ÅÇÏ´Â È¿¼Ò·Î½á, In-Fusion HD Cloning Plus CE Á¦Ç°¿¡ ÇÔ²² Á¦°øµÇ¸ç, º°µµ·Î ±¸¸Å °¡´ÉÇÕ´Ï´Ù (
Cloning Enhancer (Code 639615))
Cloning Enhancer´Â insert PCR ÁõÆø»ê¹°ÀÌ ¿ÀÁ÷ ´ÜÀÏ ¹êµå (a single PCR fragment of the expected size)·Î ÁõÆøµÇ¾úÀ» °æ¿ì¿¡¸¸ ÃßõÇϸç, ÀϹÝÀûÀ¸·Î´Â column ŸÀÔÀÇ
NucleoSpin Gel and PCR Clean-Up »ç¿ëÀ» ±ÇÀåÇÕ´Ï´Ù.
[In-Fusion Cloning¿ë PCR primer µðÀÚÀÎ °ü·Ã]
Q7. Vector¿Í insert ¸»´ÜÀÇ 15bp »óµ¿¼¿À» ºÎ°¡Çϱâ À§ÇÑ PCR primer´Â ¾î¶»°Ô µðÀÚÀÎÇϳª¿ä?
PCR primerÀÇ Forward primer (5' ¡æ 3' sense strand)¿Í Reverse primer (5' ¡æ 3' antisense strand)´Â °¢°¢ ¾Æ·¡¿Í °°Àº Á¶°ÇÀ» Æ÷ÇÔÇÏ¿© µðÀÚÀÎÇØ¾ß ÇÕ´Ï´Ù.
- Gene specific ºÎÀ§ PCR primer 3¡¯ ¸»´Ü- ƯÀ̼º°ú È¿À²ÀÌ ³ôÀº PCR ÁõÆøÀ» À§ÇÏ¿© ¾à 18-25 bpÀÇ template-specific ¼¿À»
Æ÷ÇÔÇÏ¿©¾ß ÇÕ´Ï´Ù. 3' ¸»´Ü ºÎÀ§´Â ¾Æ·¡ÀÇ Á¶°ÇÀ» ÃæÁ·ÇØ¾ß ÇÕ´Ï´Ù.
- ±æÀÌ: 18 - 25bp
- GC ÇÔ·®: 40 - 60%
- Melting temperature: 58 - 65¡É.
- Forward¿Í Reverse primer°£ÀÇ Tm°ª Â÷ÀÌ: 4¡É ÀÌÇÏ
* Note: Tm °ªÀº PCR primer Àüü ±æÀ̰¡ ¾Æ´Ï¶ó 3' gene-specific ¸»´Ü ºÎÀ§¸¦ ±âÁØÀ¸·Î °è»êÇÕ´Ï´Ù. ¸¸¾à Tm°ªÀÌ ³Ê¹« ³·´Ù¸é
gene-specific ºÎÀ§ÀÇ ¼¿ ±æÀ̸¦ ´Ã·Á Tm°ªÀÌ 58 - 65¡É·Î ¸ÂÃ߽ʽÿÀ.
- °°Àº ¿°±â¼¿ÀÌ Áߺ¹ ³ª¿µÇÁö ¾Êµµ·Ï À¯ÀÇÇϽʽÿÀ. °¢ PCR primerÀÇ 3' ¸»´ÜºÎÀ§´Â µÎ °³ ÀÌ»óÀÇ guanine (G) ¶Ç´Â cytosine (C)°¡ Æ÷ÇÔµÇÁö
¾Êµµ·Ï µðÀÚÀÎÇϽʽÿÀ.
- 15bp overlap ºÎÀ§ PCR primer 5¡¯ ¸»´Ü- »óµ¿ ÀçÁ¶ÇÕ ºÎÀ§ (homologous overlaps)ÀÇ ¼¿Àº 12 bp ÀÌÇÏ ¶Ç´Â 21 bp ÀÌ»óÀÇ
±æÀÌÀÇ ¼¿Àº ±ÇÀåÇÏÁö ¾Ê½À´Ï´Ù. 15 bp overlap ¼¿Àº ¹Ýµå½Ã °¢ DNA Fragment ¸»´Ü ºÎÀ§¿¡ À§Ä¡ÇÏ¿©¾ß ÇÕ´Ï´Ù.
- (Optional) Translation reading frameÀ» ¸ÂÃß¾îÁÖ±â À§ÇÏ¿©, ¶Ç´Â Á¦ÇÑÈ¿¼Ò »çÀÌÆ®¸¦ º¸Á¸Çϱâ À§ÇÏ¿© PCR primerÀÇ
template-specific ºÎÀ§¿Í 15 bp overlap ¼¿ »çÀÌ¿¡ Ãß°¡ÀûÀÎ ¿°±â¸¦ ºÎ°¡ÇÒ ¼ö ÀÖ½À´Ï´Ù
Q8. Vector¿Í InsertÀÇ »óµ¿Á¶ÇÕ ºÎÀ§ (Homologous overlap)ÀÇ ÃÖÀûÀÇ ±æÀÌ´Â?
ÃÖÀûÀÇ »óµ¿¼¿ ±æÀÌ´Â 15 bpÀ̸ç, 12 bp ÀÌÇÏ ¶Ç´Â 21 bp ÀÌ»óÀÇ ±æÀÌ´Â ±ÇÀåÇÏÁö ¾Ê½À´Ï´Ù.
Q9. In-Fusion CloningÀ» À§ÇÑ ¿Â¶óÀÎ µðÀÚÀÎ ÅøÀÌ ÀÖ½À´Ï±î?
´ÙÄ«¶ó¿¡¼´Â º¸´Ù ½¬¿î In-Fusion CloningÀÇ »ç¿ëÀ» À§ÇÏ¿©, ¾Æ·¡¿Í °°Àº ÅøÀ» Á¦°øÇÕ´Ï´Ù.
- Primer Design Tool: º» µðÀÚÀÎÅøÀ» ÀÌ¿ëÇϸé single cloning, multi-fragment cloning »Ó¸¸ ¾Æ´Ï¶ó, mutagenesis¸¦ À§ÇÑ In-Fusion Cloning¿ë PCR primer¸¦ Á¦ÀÛ °¡´ÉÇÕ´Ï´Ù.
º» ¿Â¶óÀÎ ÅøÀº Mozilla Firefox ¶Ç´Â Google Chrome Web browser¿¡ ÃÖÀûÈ µÇ¾îÀÖÀ¸¸ç, Internet Explorer´Â ±ÇÀåÇÏÁö ¾Ê½À´Ï´Ù.
- Molar Ratio Calculator: In-Fusion Cloning ¹ÝÀÀ¿¡ ÃÖÀû Á¶°ÇÀÎ vector, insert molar ratio¸¦ °è»êÇØÁÖ´Â ÅøÀÔ´Ï´Ù
- SnapGene Veiwer: In-Fusion CloningÀÇ ¿¹»óµÇ´Â ¹ÝÀÀ »ê¹°À» ½Ã°¢ÈÇÏ¿© º¸¿©ÁÝ´Ï´Ù.
Q10. In-Fusion Cloning ¹ÝÀÀ ½Ã¿¡, reading frameÀº ¾î¶»°Ô ¸ÂÃçÁÖ³ª¿ä?
Reading frameÀº PCR primer ¼¿ µðÀÚÀÎ °úÁ¤¿¡¼ ¸ÂÃçÁÙ ¼ö ÀÖ½À´Ï´Ù. ¿¹¸¦ µé¾î, In-Fusion PCR primer 5' ¸»´Ü ºÎÀ§ÀÇ 15 bp overlap ¼¿ÀÌ vector reading frameÀÇ 5°³ÀÇ ÄÚµ·¿¡
»óÀÀÇÑ´Ù¸é, »õ·Î¿î À¯ÀüÀÚ³ª tag¸¦ 3' ¸»´ÜÀÇ »óµ¿¼¿ ºÎÀ§¿¡ ±×´ë·Î ¿¬°á½ÃÄÑ º¤ÅÍÀÇ C-terminusÀÇ °°Àº reading frame downstream¿¡ Ŭ·Î´×ÇÒ ¼ö ÀÖ½À´Ï´Ù. Reading frameÀ»
¸ÂÃçÁÖ±â À§ÇØ target gene ºÎÀ§ÀÇ Ã¹ ¹øÂ°, ¶Ç´Â 15 bp »óµ¿¼¿ µÚÂÊ¿¡ Çϳª ¶Ç´Â µÎ °³ÀÇ ¿°±â¸¦ Ãß°¡ÇØ ÁÙ ¼ö ÀÖ½À´Ï´Ù.
<¿¹½Ã>
Q11. In-Fusion PCR primer¿¡ small external sequence¸¦ Ãß°¡ÇÒ ¼ö ÀÖ³ª¿ä?
¿¹, Ãß°¡ÇÒ ¼ö ÀÖ½À´Ï´Ù. Small tags, kozak sequence, restriction site¿Í °°Àº external nucleotide sequence¸¦ In-Fusion PCR primerÀÇ 15 bp »óµ¿¼¿°ú gene-specific
ºÎÀ§ »çÀÌ¿¡ Ãß°¡ÇÒ ¼ö ÀÖ½À´Ï´Ù. ÀÚ¼¼ÇÑ ³»¿ëÀº
In-Fusion Cloning Webinar ½Ã¸®Á Âü°í ÇϽʽÿÀ.
[In-Fusion Cloning ½ÇÇè °ü·Ã FAQ]
Q12. In-Fusion Cloning ½Ã, Insert PCRÀ» À§ÇÏ¿© ÃßõÇÏ´Â PCR È¿¼Ò´Â ¹«¾ùÀԴϱî?
PCR ¹ÝÀÀ »ê¹°ÀÇ 3¡¯ overhangÀº In-Fusion Cloning ¹ÝÀÀ¿¡ ¿µÇâÀ» ÁÖÁö ¾Ê±â ¶§¹®¿¡, ½ÃÁßÀÇ ¾î¶°ÇÑ PCR È¿¼Òµµ »ç¿ë °¡´ÉÇÕ´Ï´Ù. ÇÏÁö¸¸, PCR ¹ÝÀÀ ½ÃÀÇ ¿°±â¼¿
¿¡·¯ ¹ß»ýÀ» ÃÖ¼ÒÈÇϱâ À§ÇÏ¿©
CloneAmp HiFi PCR Premix (Code 639298)°ú
°°Àº high-fidelity PCR È¿¼ÒÀÇ »ç¿ëÀ» ±ÇÀåÇÕ´Ï´Ù. º» È¿¼Ò´Â ³ôÀº Á¤È®µµ¿Í ÁõÆø·ÂÀ¸·Î ¾à 6 kb Human genomic DNA, 10kb
E.coli genomic DNA, 15kb Lamda DNA±îÁö
ÁõÆøÀÌ °¡´ÉÇϸç, 2-step, 3-step PCR ¹ÝÀÀÀ¸·Î Àû¿ë °¡´ÉÇÕ´Ï´Ù.
* 10 kb ÀÌ»óÀÇ ÁõÆø¿¡´Â PrimeSTAR¢ç GXL DNA Polymerase (Code R050A)¸¦ ÃßõÇÕ´Ï´Ù.
±×¸². Mutation frequency of CloneAmp HiFi Polymerase compared to other high-fidelity PCR enzymes.
Q13. ÇѹøÀÇ In-Fusion Cloning ¹ÝÀÀÀ¸·Î ¿©·¯ °³ÀÇ insert¸¦ »ðÀÔÇÒ ¼ö ÀÖ³ª¿ä? (Multiple Cloning)
¿¹. ´Ù¼¸ °³ÀÇ insert fragment¸¦ Çѹø¿¡ Ŭ·Î´×ÇÑ »ç·Ê°¡ ÀÖ½À´Ï´Ù.
¾Æ·¡ÀÇ ±×¸²°ú °°ÀÌ µÎ °³ÀÇ insert »çÀÌ¿¡´Â ÇϳªÀÇ »óµ¿ÀçÁ¶ÇÕ ºÎÀ§ (homologous overlap)°¡ À§Ä¡ÇÏ¿©¾ß ÇÕ´Ï´Ù. Multiple fragment cloning (ÀÌÇÏ, Multiple cloning)À» À§ÇÑ PCR primer´Â
¿Â¶óÀÎ ÇÁ¶óÀÌ¸Ó µðÀÚÀÎÅøÀ» ÀÌ¿ëÇÏ¿´À¸¸ç, ÀÌ¿¡ ´ëÇÑ ÀÚ¼¼ÇÑ µðÀÚÀÎ °¡À̵å´Â
Primer Design Tutorial_Cloning Multiple Fragments into a Restriction Digest-Linearized VectorÀ» Âü°íÇϽʽÿÀ.
±×¸². The In-Fusion HD Cloning System has an improved capability for cloning multiple fragments in a single reaction.
Using this system, cloning up to four 1-kb fragments simultaneously is as easy as cloning a single fragment. This saves weeks that would otherwise be spent screening clones and subcloning.
Q14. In-Fusion Cloning ½Ã, insert¿Í vector¸¦ ¹Ýµå½Ã Á¤Á¦ÇØ¾ß Çϳª¿ä?
¿¹. PCR·Î ÁõÆøÇÑ DNA´Â Ŭ·Î´× ¹ÝÀÀ Àü¿¡ ¹Ýµå½Ã Á¤Á¦ÇØ¾ß ÇÕ´Ï´Ù.
ÁõÆø PCR »ê¹°À» Àü±â¿µµ¿À¸·Î ºÐ¸®ÇÏ¿© ¿øÇÏ´Â Å©±â¿¡¼ ´ÜÀϹêµå·Î È®ÀÎÀÌ µÇ´Â °æ¿ì, Ä÷³ ŸÀÔ Á¤Á¦Å°Æ® (NucleoSpin Gel and PCR Clean Up) ¶Ç´Â Cloning Enhancer (CE)
Áß ¼±ÅÃÇØ¼ »ç¿ëÇÒ ¼ö ÀÖ½À´Ï´Ù.
ÇÏÁö¸¸ Àü±â¿µµ¿ °á°ú ¹é±×¶ó¿îµå ¶Ç´Â ´ÙÁß¹êµå (multiple bands)°¡ °üÂûÀÌ µÇ´Â °æ¿ì, Ä÷³ ŸÀÔÀÇ Á¤Á¦ ŰƮ¸¦ ÀÌ¿ëÇÏ¿© Ÿ°Ù ´ÜÀϹêµå¸¸ ºÐ¸® Á¤Á¦Çϱ⸦ ±ÇÀåÇÕ´Ï´Ù.
ÇÏÁö¸¸, Àü±â¿µµ¿ °á°ú ¹é±×¶ó¿îµå ¶Ç´Â ´ÙÁß¹êµå (multiple bands)°¡ °üÂûÀÌ µÇ´Â °æ¿ì, Ä÷³ ŸÀÔÀÇ Á¤Á¦ ŰƮ¸¦ ÀÌ¿ëÇÏ¿© Target fragment band¸¦ ºÐ¸®Çϱ⸦ ±ÇÀåÇÕ´Ï´Ù.
- NucleoSpin Gel and PCR Clean Up (Ä÷³Å¸ÀÔ)
- PCR »ê¹°ÀÌ multiple band¸¦ º¸ÀÌ´Â °æ¿ì ¿øÇϴ Ÿ°Ù ¹êµå¸¸ gel extraction
- PCR »ê¹°ÀÌ background smear°¡ º¸ÀÌÁö ¾Ê´Â °æ¿ì¿¡´Â º» Á¦Ç°À¸·Î PCR »ê¹° Á¤Á¦µµ °¡´É
- Cloning Enhancer (CE)
- PCR ¹ÝÀÀ ½ÃÀÇ ÀÜ¿©¹°À» Á¦°ÅÇÏ´Â È¿¼Ò
- Gel»ó¿¡¼ background smear ¾øÀÌ ¿øÇÏ´Â »çÀÌÁî¿¡ single band·Î È®ÀεǴ °æ¿ì¿¡ »ç¿ë
Q15. In-Fusion Cloning¿¡ Àû¿ë °¡´ÉÇÑ Insert ÃÖ´ë Å©±â´Â ¾ó¸¶ÀԴϱî?
In-Fusion CloningÀ» ÀÌ¿ëÇÏ¿©, pUC19 vector¿¡ 15 kb insertÀÇ Å¬·Î´×À» Å×½ºÆ® ¿Ï·áÇÑ ¹Ù ÀÖ½À´Ï´Ù.
±×¸². Ten out of ten colonies contain the correct insert (100% efficiency) when cloning fragments as large as 15 kb (results confirmed by colony PCR screening).
Q16. In-Fusion Cloning¿¡ Àû¿ëÇÒ ¼ö ÀÖ´Â Insert ÃÖ¼Ò Å©±â´Â ¾ó¸¶ÀԴϱî?
In-Fusion Cloning Å×½ºÆ® °á°ú, 50 bp synthetic oligonucleotide (vector ¸»´ÜÀÇ 15bp overlap ¼¿ Æ÷ÇÔ)ÀÇ Å¬·Î´×¿¡ ¼º°øÇÑ ¹Ù ÀÖ½À´Ï´Ù. ªÀº ¼¿ÀÇ synthetic oligos (50 - 150 nt)¸¦ ÀÌ¿ëÇÏ¿© In-Fusion CloningÀ» ÁøÇàÇÒ °æ¿ì, oligo¿Í vectorÀÇ molar ratio´Â 5 - 15 : 1 ·Î ±ÇÀåÇÕ´Ï´Ù. OligoÀÇ ±æÀÌ¿¡ µû¶ó, ÃÖÀûÀÇ molar ratio´Â ´Þ¶óÁú ¼ö ÀÖ½À´Ï´Ù.
* Note: Non-phosphorylated oligonucleotides´Â In-Fusion Cloning¿¡ Àû¿ë °¡´ÉÇÕ´Ï´Ù. ÇÏÁö¸¸, In-Fusion Enzyme mixÀÇ 3¡¯ exonuclease activity¸¦
À§Çؼ´Â ¸»´ÜºÎÀ§ÀÇ 3¡¯-OH GroupÀÌ ÇÊ¿äÇÕ´Ï´Ù.
Q17. In-Fusion Cloning¿¡ Àû¿ëÇÒ ¼ö ÀÖ´Â VectorÀÇ ÃÖ´ë Å©±â´Â ¾ó¸¶ÀԴϱî?
In-Fusion CloningÀ» ÀÌ¿ëÇϸé, transfer/shuttle vector¸¦ »ç¿ëÇÏÁö ¾Ê°í Adenoviral vector (32.6 - 36 kb)¿Í °°Àº Å« »çÀÌÁîÀÇ vector¿¡µµ ½±°Ô single cloning
¶Ç´Â multiple cloningÀ» ÁøÇàÇÒ ¼ö ÀÖ½À´Ï´Ù.
Adeno-X Adenoviral System 3 ºê·Î¼ÅÀÇ Figure 1, 2, 5¿Í Table IIIÀÇ ³»¿ëÀ» ÂüÁ¶ÇϽʽÿÀ.
Q18. In-Fusion CloningÀ» ÀÌ¿ëÇÏ¿© Mutagenesis ½ÇÇè¿¡ Àû¿ëÇÒ ¼ö ÀÖ³ª¿ä?
¿¹. In-Fusion Cloning ¹ÝÀÀÀ» ÀÌ¿ëÇÏ¿© single & multiple base change (¿°±âº¯ÀÌ), deletion (Á¦°Å), insertion (»ðÀÔ) º¯ÀÌ¿¡ Àû¿ë °¡´ÉÇÕ´Ï´Ù. ´ÙÄ«¶ó¿¡¼´Â mutagenesis¸¦
À§ÇÑ
In-Fusion PCR primer µðÀÚÀÎÅøÀ» Á¦°øÇϰí ÀÖ½À´Ï´Ù.
»ó¼¼ÇÑ ³»¿ëÀº
In-Fusion Webinar ½Ã¸®Á Âü°í ÇϽʽÿÀ.
Q19. In-Fusion Cloning ¹ÝÀÀ ½Ã, ±ÇÀåµÇ´Â Insert:Vector molar ratio´Â ¹«¾ùÀԴϱî?
- ÀϹÝÀûÀÎ ½ÇÇè Á¶°Ç ¶Ç´Â single, multiple fragment cloning ½Ã, insert : vector = 2 : 1À» ±ÇÀåÇÕ´Ï´Ù.
- Linearized, purified vector¿¡ ´ëÇÏ¿© °¢°¢ÀÇ insert fragmentÀÇ molar ratio ºñÀ²Àº 2 : 1ÀÌ µÇ¾î¾ß ÇÕ´Ï´Ù. ¿¹¸¦ µé¾î, µÎ °³ÀÇ insert cloningÀÏ °æ¿ì,
insert A : insert B : vector = 2 : 2 : 1 ±ÇÀå
- ÀÛÀº »çÀÌÁîÀÇ DNA Fragments (150 bp - 350 bp)¸¦ ÀÌ¿ëÇÏ´Â °æ¿ì, insert : vector = 3 - 5 : 1 ±ÇÀå
- ªÀº ±æÀÌÀÇ ÇÕ¼º ¿Ã¸®°í (50 bp - 150 bp)¸¦ ÀÌ¿ëÇÏ´Â °æ¿ì, insert : vector = 5 - 15 : 1 ±ÇÀå. ÇÏÁö¸¸, Oligo ±æÀÌ¿¡ µû¶ó¼ ÃÖÀûÀÇ molar ratio ±æÀÌ´Â ´Þ¶óÁú ¼ö ÀÖ½À´Ï´Ù.
- In-Fusion Cloning ¹ÝÀÀ ½ÃÀÇ Molar ratio °è»êÀº Molar Ratio Calculator ¿Â¶óÀÎÅø¸¦ ÀÌ¿ëÇϽʽÿÀ.
Q20. In-Fusion enzyme Incubation ½Ã°£À» ±æ°Ô Çϸé Ŭ·Î´× È¿À²À» Áõ°¡½Ãų ¼ö ÀÖ³ª¿ä?
In-Fusion ¹ÝÀÀ ½Ã°£À» ÀÓÀÇ·Î ´Ã¸®´Â °ÍÀº ±ÇÀåÇÏÁö ¾Ê½À´Ï´Ù. ÀÌ´Â ÀǵµÄ¡ ¾ÊÀº vector¿Í insert ¸»´Ü »çÀÌÀÇ single-strand ºÎÀ§¸¦ ¹ß»ý½Ãų ¼ö ÀÖÀ¸¸ç,
»óµ¿Á¶ÇÕÀÇ È¿À²À» ¶³¾î¶ß¸± »Ó¸¸ ¾Æ´Ï¶ó ÀüüÀûÀΠŬ·Î´× È¿À²À» ÀúÇϽÃŵ´Ï´Ù. ¸Å´º¾ó¿¡¼ ±ÇÀåÇÏ´Â ½Ã°£À» ÁؼöÇϼ¼¿ä.
Q21. In-Fusion Cloning¿¡ ±ÇÀåµÇ´Â Comp.cellÀÇ Á¾·ù´Â ¹«¾ùÀԴϱî?
In-Fusion Cloning »ç¿ë ½Ã, ÃÖ¼Ò 10
8 cfu/§¶ supercoiled DNA ÀÌ»óÀÇ È¿À²À» °¡Áø bacterial cell »ç¿ëÀ» ±ÇÀåÇÕ´Ï´Ù.
- Stellar Competent Cell (Code 636763) (ÀϺΠIn-Fusion Cloning Kit Á¦Ç° ³» Æ÷ÇÔ)Àº In-Fusion Cloning¿¡ ÃÖÀûÈ µÇ¾îÀÖÀ» »Ó¸¸ ¾Æ´Ï¶ó, ÀϹÝÀûÀΠŬ·Î´× ¿ëµµ·Î ÀÌ¿ë °¡´ÉÇÕ´Ï´Ù.
- Stellar Competent CellÀº BACs, fosmid¿Í °°Àº Å« »çÀÌÁîÀÇ º¤ÅÍÀÇ ÁõÆø°ú Ŭ·Î´×¿¡ validation µÇ¾îÀÖÀ» »Ó¸¸ ¾Æ´Ï¶ó, Retrovirus/LentivirusÀÇ LTR, AdenovirusÀÇ ITR ºÎÀ§¿Í °°Àº ¹Ýº¹µÇ´Â ¼¿À» °¡Áø º¤ÅÍÀÇ Å¬·Î´×¿¡µµ »ç¿ë °¡´ÉÇÕ´Ï´Ù.
- TOP10 cell ȤÀº ±× À¯µµ¼¼±Õ (¿¹¸¦ µé¾î, ccdB Survavial 2T1R E.coli), ¶Ç´Â ¿¬°ü ¹ÚÅ׸®¾Æ (¿¹¸¦ µé¾î, DH10B, MC1061)ÀÇ °æ¿ì´Â Recombinant cloneÀÇ »ý¼º·üÀÌ ºñ±³Àû Àû´Ù°í ¾Ë·ÁÁ® ÀÖÀ¸¹Ç·Î, multiple-fragment cloning ¶Ç´Â low copy vector¸¦ ÀÌ¿ëÇÑ cloning ½ÇÇè¿¡´Â ±ÇÀåÇÏÁö ¾Ê½À´Ï´Ù.
Q22. Cloning ¹ÝÀÀ »ê¹°À» ÇüÁúÀüȯ ½Ã¿¡, ¸Å´º¾ó¿¡¼ ±ÇÀåÇÏ´Â ¾çº¸´Ù ´õ ¸¹ÀÌ TransformationÇØµµ µÇ³ª¿ä?
±ÇÀåÇÏÁö ¾Ê½À´Ï´Ù. User manual »ó¿¡¼ ±ÇÀåÇÏ´Â ¾ç ÀÌ»óÀ» »ç¿ëÇÒ °æ¿ì, cell¿¡ µ¶¼ºÀ» ³ªÅ¸³¾ ¼ö ÀÖ½À´Ï´Ù. In-Fusion Cloning ¹ÝÀÀ »ê¹°À» ÇüÁú Àüȯ ½Ã,
Stellar Competent Cell 50 §¡ ´ç, 2.5 §¡ »ç¿ëÇϱ⸦ ±ÇÀåÇÕ´Ï´Ù.