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Team:Hannover/Protocols/Cloning/Ligation
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| - | <h1><a href="https://2014.igem.org/Team:Hannover/Protocols | + | <h1><a href="https://2014.igem.org/Team:Hannover/Protocols">Protocols</a> / Ligation </h1> |
<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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<tr><td><b>Volume [μl]</b></td><td><b>Compounds of the digest</b></td></tr> | <tr><td><b>Volume [μl]</b></td><td><b>Compounds of the digest</b></td></tr> | ||
<tr><td>2.00</td><td>10 x T4 DNA Ligase buffer </td></tr> | <tr><td>2.00</td><td>10 x T4 DNA Ligase buffer </td></tr> | ||
| - | <tr><td>2.00</td><td>10 mM | + | <tr><td>2.00</td><td>10 mM ATP</td></tr> |
<tr><td>1.00</td><td>1 T4 DNA Ligase</td></tr> | <tr><td>1.00</td><td>1 T4 DNA Ligase</td></tr> | ||
<tr><td> -</td><td>20-100 ng linear vector DNA</td></tr> | <tr><td> -</td><td>20-100 ng linear vector DNA</td></tr> | ||
<tr><td> -</td><td>100-500 ng insert DNA</td></tr> | <tr><td> -</td><td>100-500 ng insert DNA</td></tr> | ||
| - | <tr><td colspan="2">ad 20 µl | + | <tr><td colspan="2">ad 20 µl H<sub>2</sub>O</td></tr></table></td><td><table colspan="2" ><tr><td><b>Cycler Program</b></td></tr><tr><td>Step</td><td>Temperature [°C]</td><td>Time [min]</td><td>Cycle no.</td></tr><tr><td>1.</td><td>22</td><td>60.0</td><td>1</td></tr><tr><td>2.</td><td>80</td><td>10.0</td><td>1</td></tr></table></td></tr></table> |
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Latest revision as of 23:35, 17 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.
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