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Team:INSA-Lyon/Protocols/Ligation
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(New page: ===Ligation=== <html lang="en"> <ul> <br><br> <span style="font-style : italic">If it follows a digestion without purification: <u>thermoinactivation 20 min at 70°C </u></span> <br><br>...) |
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<span style="font-style : italic">If it follows a digestion without purification: <u>thermoinactivation 20 min at 70°C </u></span> | <span style="font-style : italic">If it follows a digestion without purification: <u>thermoinactivation 20 min at 70°C </u></span> | ||
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<li>Add 0,5 µL T4 DNA ligase (0,5 U) --> Add the enzyme last</li> | <li>Add 0,5 µL T4 DNA ligase (0,5 U) --> Add the enzyme last</li> | ||
<li>Add sterile water qs 20 µL</li> | <li>Add sterile water qs 20 µL</li> | ||
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<I>Incubate 3h at room temperature or overnight at 15°C </I> | <I>Incubate 3h at room temperature or overnight at 15°C </I> | ||
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Revision as of 10:01, 19 August 2010
Ligation
- Add x µL DNA (the entire digestion gel purification for part ligation) Plasmid: 50 to 200 ng; Insert: 100 ng (0,5 kb) to 1 µg (10 kb))
- Add 2 µL Buffer T4 DNA ligase (Attention: this buffer contains ATP, defrost on ice)
- Add 0,5 µL T4 DNA ligase (0,5 U) --> Add the enzyme last
- Add sterile water qs 20 µL
If it follows a digestion without purification: thermoinactivation 20 min at 70°C
Incubate 3h at room temperature or overnight at 15°C
