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Total 34 articles, 2 pages/ current page is 1
  Total : 34, 1 / 2 pages
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  ±è¼ö¿¬ 2016-03-08 16:11:06, Hit : 4211
Á¦ ¸ñ   shRNA Á¦ÀÛ ÇÁ·ÎÅäÄÝ
[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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