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ÀüÁ¦Ç°º¸±â
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Cloning °ü·Ã
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¼ö½ÄÈ¿¼Ò£¯Nuclease¡¢Topoisomerase
> DNA Topoisomerase I
DNA Topoisomerase I
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Takara
2240A
DNA Topoisomerase I
100 U
84,800¿ø
106,000¿ø
03.02 ~ 04.30
Takara
2240B
DNA Topoisomerase I
500 U
477,000¿ø
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Yatalase (°è»ó±Õ ¼¼Æ÷..
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Çü»ó
20 mM
Potassium Phosphate (pH7.2)
50 mM
KCl
0.05 mM
EDTA
5 mM
2- Mercaptoethanol
0.02%
BSA
50%
Glycerol
º¸Á¸ -20¡É
÷ºÎ Buffer Á¶¼º(10¡¿)
350 mM
Tris-HCl (pH8.0)
720 mM
KCl
50 mM
MgCl
2
50 mM
DTT
50 mM
Spermidine
0.1%
BSA(º°Ã·)
±â¿ø
Calf thymus
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º» È¿¼Ò´Â ±×¸² 1¿¡ ³ªÅ¸³½ 4Á¾·ùÀÇ ¹ÝÀÀÀ» Ã˸ÅÇÏ´Â È¿¼ÒÀÌ´Ù. ¼Û¾ÆÁö Èä¼± À¯·¡À̹ǷΠ¿øÇÙ »ý¹° À¯·¡È¿¼Ò¿Í ¼ºÁúÀÌ ´Þ¶ó, Mg2£«°¡ Á¸ÀçÇÏÁö ¾Ê¾Æµµ Ȱ¼ºÀ» ³ªÅ¸³½´Ù. ¶Ç ¿øÇÙ »ý¹°ÀÇ DNA Topoisomerase IÀÌ (-)ÀÇ supercoil ºÐÀÚ¿¡¸¸ ÀÛ¿ëÇϴµ¥ ¹ÝÇØ º» È¿¼Ò´Â (+), (-)ÀÇ supercoil ºÐÀÚ¸¦ Ç®¼ö ÀÖ´Ù(±×¸² 1).
±×¸² DNA Topoisomerase IÀÌ Ã˸ÅÇÏ´Â ¹ÝÀÀ
1. DNAÀÇ supercoil »óŸ¦ ÀÌ¿Ï
2. ssDNA ºÐÀÚ ³»¿¡ DNA °¡´ÚÀÇ ¸ÅµìÀ» ¸¸µé°Å³ª(knotting), ¿ªÇüÅ·ÎÀÇ ¹ÝÀÀ (unknotting)
3. »óº¸ÀûÀÎ ¿°±â¼¿À» °®´Â ssDNA·ÎºÎÅÍ ds closed circular DNA ºÐÀÚ¸¦ Çü¼ºÇÏ´Â ¹ÝÀÀ
4. µÎ ds circular DNA ºÐÀÚÀÇ ¾î´À ÇÑÂÊ¿¡ nickÀÌ Á¸ÀçÇÒ °æ¿ì µÎ ºÐÀÚÀÇ ¿¬°á¹ÝÀÀ
(catenation) ¶Ç´Â ¿ªÀÇ ¹ÝÀÀ(decatenation)À» Ã˸Å
Ȱ¼ºÀÇ Á¤ÀÇ
0.5 §¶/50 §¡ÀÇ supercoiled pBR322 DNA¸¦ 37¡É¿¡¼ 30ºÐ µ¿¾È 100% relaxed formÀ¸·Î Àüȯ½ÃŰ´Â È¿¼ÒȰ¼ºÀ» 1 UÀ¸·Î ÇÑ´Ù.
Ȱ¼º ÃøÁ¤¿ë ¹ÝÀÀ¾× Á¶¼º
35 mM
Tris-HCl(pH8.0)
72 mM
KCl
5 mM
MgCl
2
5 mM
DTT
5 mM
Spermidine
0.01%
BSA
0.5 §¶/50 §¡
Supercoiled pBR322 DNA
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24 UÀÇ º» È¿¼Ò¿Í 0.5 §¶ÀÇ supercoiled pBR322 DNA¸¦ 37¡É, 24½Ã°£ ¹ÝÀÀÇÏ¿©µµ DNAÀÇ EtBr Á¸ÀçÇÏ¿¡¼ Àü±â¿µµ¿ ÆÐÅÏ¿¡ º¯È°¡ ÀϾÁö ¾Ê´Â´Ù.
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- DNA conformationÀÇ º¯È¯°ú ÇØ¼®
»ç¿ë»óÀÇ ÁÖÀÇ
- ÷ºÎ¹ÝÀÀ¾×¿¡´Â spermidine (ÃÖÁ¾³óµµ 5 mM)ÀÌ Ã·°¡µÇ¾î ÀÖ´Ù. SpermidineÀ» ÷°¡ÇÏÁö ¾Ê´Â °æ¿ì Ȱ¼ºÀÌ 1/20Á¤µµ·Î ÀúÇÏÇÏ´Â ¼ö°¡ ÀÖ´Ù. Spermidine À» ÷°¡ÇÒ ¶§´Â HCI·Î pH8.0·Î Á¶Á¤ÇÏ¿© »ç¿ëÇÒ °Í.
- pBR322 DNA¿Í ¬æX174 DNAÀÇ RF I (supercoil ºÐÀÚ)À» DNA Topoisomerase IÀ¸·Î ¹ÝÀÀÇÑ ÈÄ, Àü±â¿µµ¿Çϸé gelÁß¿¡ EtBrÀÌ Æ÷ÇԵǾî ÀÖ´Â °æ¿ì ¹êµåÀÇ À§Ä¡¿¡ º¯È°¡ ÀϾÁö ¾Ê´Â´Ù.
À̰ÍÀº DNA Topoisomerase I¿¡ ÀÇÇØ relaxed formÀ¸·Î º¯È¯ÇÑ DNA°¡ EtBrÀÇ ¿µÇâÀ¸·Î ´Ù½Ã supercoil »óÅ·Πº¯Çعö¸®±â ¶§¹®ÀÌ´Ù.
µû¶ó¼ DNA Topoisomerase IȰ¼ºÀ» ÃøÁ¤ÇÒ ¶§´Â EtBrÀÌ Æ÷ÇÔµÇÁö ¾ÊÀº ¾Æ°¡·Î½º °Ö¿¡¼ Àü±â¿µµ¿À» ½Ç½ÃÇÑ ´ÙÀ½ EtBr·Î ¿°»öÇÒ Çʿ䰡 ÀÖ´Ù.
- 10¡¿ ÷ºÎ ¹öÆÛ¿¡ 0.1% BSA¸¦ Á÷Á¢ ÷°¡ÇÏ¸é ´Ù·®ÀÇ ¹é»ö ħÀüÀÌ »ý±â¹Ç·Î ¹ÝÀÀ¾×À» Á¶Á¦ÇÒ ¶§´Â ´ÙÀ½ÀÇ ¼ø¼·Î ÇÑ´Ù.
dH
2
O ¡æ 10¡¿ ÷ºÎ ¹öÆÛ ¡æ 0.1% BSA ¡æ ±âÁú DNA
- º» È¿¼Ò´Â À¯¸®³ª ÇÃ¶ó½ºÆ½¿¡ ´ëÇÏ¿© ÈíÂø¼ºÀÌ Å©¹Ç·Î ¹ÝÀÀÇÒ ¶§´Â BSA(Bovine Serum Albumin)À» ÷°¡ÇÏ´Â °ÍÀÌ ¹Ù¶÷Á÷ÇÏ´Ù.
Keyword :
¼ö½ÄÈ¿¼Ò£¯Nuclease¡¢Topoisomerase
BAL 31 Nuclease
DNA Topoisomerase I
Cloned
Exonuclease I
Cloned
Exonuclease III
Micrococcal Nuclease
Mung Bean Nuclease
RT-PCR Çʼöǰ
Recombinant DNase I (RNase-free)
Cloned
Ribonuclease H (RNase H)
S1 Nuclease