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Total 34 articles, 2 pages/ current page is 1 |
Total : 34, 1 / 2 pages
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2016-03-08 16:11:06, Hit : 4211
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Á¦ ¸ñ |
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shRNA Á¦ÀÛ ÇÁ·ÎÅäÄÝ |
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[¹æ¹ý 1]
1) Vector (pSilencer 3.1-H1 neo vector) 3ugÀ» BamH I°ú Hind III·Î Enzyme cuttingÇÑ´Ù. (Enzyme cutting µ¿¾È 2-4 ÁøÇà)
2) FP/RP annealing – a³ª b Áß Çϳª¸¦ ¼±ÅÃÇÏ¿© mixture¸¦ ¸¸µç´Ù.
a.
FP 2 ug
RP 2 ug
10X Taq buffer 5 ul
D.W x ul
-------------------------------
Total 50 ul
b.
FP 2 ug
RP 2 ug
D.W x ul
-------------------------------
Total 50 ul
3) Incubation – 90¡É, 3 min
4) Incubaion – 37¡É, 1 hr
5) 4¹ø productÀÇ 5ul¸¦ D.W 45ul¿Í ¼¯´Â´Ù.
6) Vector gel purification
7) ligation
- (AnnealingÇÑ product(5)) + (purificationÀ» ÅëÇØ ¾òÀº vector(6))
Insert 1 ul
pSilencer (50ug/1ul) 1 ul
10X T4 ligase buffer 1 ul
T4 ligase 1 ul
D.W 6 ul
--------------------------------
Total 10 ul
8) Transformation
9) Mini-prep ÈÄ construct È®ÀÎ
[¹æ¹ý 2]
1) Vector (pSilencer 3.1-H1 neo vector) 3ugÀ» BamH I°ú Hind III·Î Enzyme cuttingÇÑ´Ù. (Enzyme cutting µ¿¾È 2-4 ÁøÇà)
2) FP/RP annealing
FP 2 ul
RP 2 ul
10X Taq buffer 5 ul
D.W 41 ul
-------------------------------
Total 50 ul
3) Incubation – 95¡É, 4 min
4) Incubaion – 70¡É, 10 min
5) AnnelaingÇÑ mixture¸¦ BamH I°ú Hind III·Î Enzyme cutting -> »ý·«°¡´É
6) Enzyme cuttingÇÑ vector¿Í annealingÇÑ mixture¸¦ gel purificationÇÑ´Ù.
- AnealingÇÑ mixture¸¦ gel loadingÇÑ ÈÄ À§¿¡ ¹êµå¸¦ À߶ó purificationÇؾßÇÑ´Ù.
- 5)¸¦ »ý·«Çß´Ù¸é annealingÇÑ mixture´Â gel purification ÇÏÁö¾Ê°í ¹Ù·Î ligation¿¡ »ç¿ëÇÑ´Ù.
6) Ligation
(Insert size:- 60 bp, Vector size - 4285 bp)
7) Transformation
8) Mini-prep ÈÄ construct È®ÀÎ
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