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Team:INSA Toulouse/contenu/lab practice/notebook/protocols/ligation
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· Add equimolar amount of XbaI PstI digested fragment (< 3 µl) <br> | · Add equimolar amount of XbaI PstI digested fragment (< 3 µl) <br> | ||
· Add 1 µl T4 DNA ligase buffer. <i><b>Note</b>: Do not use quick ligase</i><br> | · Add 1 µl T4 DNA ligase buffer. <i><b>Note</b>: Do not use quick ligase</i><br> | ||
| - | · Add 0.5 | + | · Add 0.5 µl T4 DNA ligase<br> |
| - | · Add water to 10 | + | · Add water to 10 µl<br> |
· Ligate 16C/30 min, heat kill 80C/20 min<br> | · Ligate 16C/30 min, heat kill 80C/20 min<br> | ||
| - | · Transform with 1-2 | + | · Transform with 1-2 µl of product<br> |
<i><b>Note</b>: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.</i> | <i><b>Note</b>: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.</i> | ||
</p> | </p> | ||
Latest revision as of 15:09, 3 September 2013
Notebook
Protocols
Ligation
After following our restriction digest protocol (which uses 250ng of DNA) you may follow these steps for ligation.
· Add 2µl of digested plasmid backbone (25 ng)
· Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 µl)
· Add equimolar amount of XbaI PstI digested fragment (< 3 µl)
· Add 1 µl T4 DNA ligase buffer. Note: Do not use quick ligase
· Add 0.5 µl T4 DNA ligase
· Add water to 10 µl
· Ligate 16C/30 min, heat kill 80C/20 min
· Transform with 1-2 µl of product
Note: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.
