[求救] 有人有用過T4 DNA polymerase嗎？
我是使用NEB的T4 DNA polymerase
照NEB網頁提供的protocal做 (如下)
http://www.neb.com/nebecomm/products/productM0203.asp
Usage notes:
Incubate at temperature suggested for specific protocol. Refer to specific
protocol to determine recommended dNTP concentrations.
T4 DNA Polymerase is active in any 1X NEB retriction enzyme reaction buffer
and T4 DNA Ligase reaction buffer when supplemented with dNTPs.
Application notes:
Protocol for blunting ends by 3′ overhang removal and 3′ recessed end
fill-in:
DNA should be dissolved in any 1X restriction enzyme reaction NEBuffer or 1X
T4 DNA Polymerase Reaction Buffer supplemented with 100 μM dNTPs. Add 1 unit
T4 DNA Polymerase per microgram DNA and incubate 15 minutes at 12°C. Stop
reaction by adding EDTA to a final concentration of 10 mM and heating to 75°
C for 20 minutes (1,2). CAUTION: Elevated temperatures, excessive amounts of
enzyme, failure to supplement with dNTPs or long reaction times may result in
recessed ends due to the 3′→ 5′ exonuclease activity of the enzyme
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T4 DNA polymerase可以讓Sticky end平滑化(blunting)
(有兩個不同的sticky end想要用blunting方式)
之後想ligation起來(T4 DNA ligase)
可是並沒有成功
想請問各位大大有誰用過的經驗或撇步
提供給我參考看看吧
謝謝^^
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06/07 18:13, , 1F
06/07 18:13, 1F
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