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- 文献和实验
- 技术资料
- 英文名:
FastDigest PvuI
- 规格:
20 reactions
描述
| 5' | C | G | A | T ↓ | C | G | 3' | ||||
| 3' | G | C ↑ | T | A | G | C | 5' |
Thermo Scientific FastDigest PvuI restriction enzyme recognizes CGAT^CG site and cuts best at 37°C in 5–15 minutes using universal FastDigest Buffer. Isoschizomers: BpvUI, MvrI, Ple19I.
Thermo Scientific FastDigest PvuI is one of an advanced line of fast restriction enzymes that are all 100% active in the universal FastDigest and FastDigest Green reaction buffers.
The universal buffer allows rapid single-, double-, or multiple DNA digestion within 5–15 minutes eliminating any need for buffer change or subsequent DNA clean-up steps. See Reaction Conditions for FastDigest Enzymes for a table of enzyme activity, heat inactivation and incubation times for this and other FastDigest restriction enzymes. DNA modifying enzymes, such as Klenow Fragment, T4 DNA Ligase, alkaline phosphatases and T4 DNA Polymerase all have 100% activity in FastDigest Buffer. Therefore, enzymes for downstream applications can be directly added to the FastDigest reaction mix.
For additional convenience FastDigest Green Buffer includes a density reagent and two tracking dyes for direct loading of digestion reaction products on gels.
Short incubation times and optimal composition of the universal FastDigest Buffer eliminate star activity effects.
Features
• 100% activity of all FastDigest enzymes in the universal buffer
• 100% buffer compatibility with downstream applications
• Complete digestion in 5–15 minutes
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文献和实验性,在胡萝卜II型转化酶基因启动子的克隆方面取得了新的进展。首先将胡萝卜基因组DNA 分别用PvuI、SmaI、DraI、EcoRV酶切,并设计了1个衔接头长链序列和1个衔接头短链序列,并在衔接头短链的3'末端带有1个氨基的衔接头,能够阻止聚合酶催化的衔接头短链的延伸,同时衔接头的长链和短链之间是反向重复序列。将酶切片段与此衔接头连接,取连接产物做模板,以衔接头引物和基因特异引物做PCR ,在首轮PCR 中只有限定的远端基因特异引物有结合位点,当基因特异引物延伸产生的DNA 链通过衔接头时,才能产生衔
GR/CGYC PsrI GAA***NNNNTA/C BsgI GTGCAG,16,14 PvuI CGAT/CG BsiEI CGRY/CG RleAI CCCACA,12,9 BsiWI C/GTACG RsrII CG/GWCCG BsmBI C/GTCTC SacII CCGC/GG BsrBI CCGCTC,-3,-3 SalI G/TCGAC BsrFI R/CCGGY SanDI GG/GWCCC BsrGI T/GTACA SapI G/CTCTTC BssHII
, PvuI , SacI , SacII , SalI , ScaI , SmaI , SpeI , SphI , StuI , XbaI , XhoI , XmaI ,为什么要添加保护碱基?在分子克隆实验中,有时我们会在待扩增的目的基因片段两端加上特定的酶切位点,用于后续的酶切和连接反应。由于直接暴露在末端的酶切位点不容易直接被限制性核酸内切酶切开,因此在设计 PCR 引物时,人为的在酶切位点序列的 5‘ 端外侧添加额外的碱基序列,即保护
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