Template:Team:Groningen/CONTENT/LOGBOOK/tasA Ligation t15
tasA Ligation in RFP Backbone (t15)
The ligation of tasA in the RFP backbone was performed to afterwards be able to transform it into E. coli.
Our goal is to overexpress TasA by adding a second copy of the tasA gene under the control of a salt inducible promoter. This overexpression will lead to a stronger biofilm when the biofilm is exposed to salt water.
tasA was ligated into the RFP backbone.
<a class="postscriptum protocol" href="https://2015.igem.org/Team:Groningen/Protocols_and_Protocols/Ligation">Ligation</a>
00:00, 21 May 2015 - 00:00, 21 May 2015
The RFP backbone DNA was extracted from the gel with the PCR purification kit. For this binding buffer was added to the cut out gel in the ratio 2:1 binding buffer:gel weight. It was stored as BB1, purified backbone (50 µL elution buffer). Afterwards the ligation of tasA in BB1 was carried out. For that the following components were combined.
15 µL BB1
2 µL 10x buffer (2.1)
1 µL tasA DNA (4.2)
2 µL T4 ligase
The mixture was incubated at room temperature for 2 hours. During this time ampicillin plates were poured (100 µg/mL).