Difference between revisions of "Template:Team:TU Eindhoven/Protocols HTML"
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Revision as of 16:41, 24 August 2015
Protocols
For the labwork various protocols were created. These are applied during the work in the Biolab.
General Protocols
- Preparation of general necessities
- Digestion
- Ligation
- PCR Amplification
- Small Culturing
- Transformation into NovaBlue
- Colony PCR
Gibson Assembly
Gibson Assembly is a one-pot assembly method which requires multiple dsDNA fragments as well as a linearized vector. The dsDNA fragments may be generated through PCR or be directly ordered in the form of gBlocks. The vector can either be linearized through PCR or through restriction enzyme digestion. In our case, we ordered gBlocks to obtain the dsDNA fragments and we generated the linear vector through PCR. Therefore, we use gBlocks and vector linearization in our protocols. Another important protocol for Gibson Assembly which is not listed below is Colony PCR.
- Vector Linearization - A linear vector is a prerequisite for Gibson Assembly. Linearization can be realized through restriction or through PCR. In our protocol, we use PCR as this yields scarless constructs. This protocol consists of a PCR step, an optional DpnI digestion step, an optional PCR purification step, a NanoDrop step and an optional gel electrophoresis step.
- Gibson Assembly