Team:Hannover/Protocols/Cloning/Ligation

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<p class="text">After restriction enzyme cleavage, compatible ends of two fragments can hybridize, but are not stable due to their missing phosphodiester bonds. To fix those nicks in the double-stranded DNA, a ligase from T4 phages (Thermo Scientific) was utilized. The ligation reaction mix is shown in the following table.  </p>
<p class="text">After restriction enzyme cleavage, compatible ends of two fragments can hybridize, but are not stable due to their missing phosphodiester bonds. To fix those nicks in the double-stranded DNA, a ligase from T4 phages (Thermo Scientific) was utilized. The ligation reaction mix is shown in the following table.  </p>
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<h2>Table 1: Reaction Mixes and Temperature Programs for the Ligation.</h2>
<h2>Table 1: Reaction Mixes and Temperature Programs for the Ligation.</h2>
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<table align="line" width="800px"><tr><td><table>

Revision as of 19:15, 15 October 2014

Protocols / Ligation

After restriction enzyme cleavage, compatible ends of two fragments can hybridize, but are not stable due to their missing phosphodiester bonds. To fix those nicks in the double-stranded DNA, a ligase from T4 phages (Thermo Scientific) was utilized. The ligation reaction mix is shown in the following table.



Table 1: Reaction Mixes and Temperature Programs for the Ligation.

Volume [μl]Compounds of the digest
2.0010 x T4 DNA Ligase buffer
2.0010 mM µl-1 ATP
1.001 T4 DNA Ligase
-20-100 ng linear vector DNA
-100-500 ng insert DNA
ad 20 µl H2O
Cylcer Program
StepTemperature [°C]Time [min]Cycle no.
1.2260.01
2.8010.01