Template:Team:TU Eindhoven/Protocols HTML
Protocols
For the labwork various protocols were created. These are applied during the work in the Biolab.
General Protocols
- Preparation of general necessities
- PCR Amplification
- Small Culturing
- Transformation into NovaBlue
- Colony PCR
Traditional Cloning & BioBricking
Traditional cloning remains the workhorse of DNA recombinant technology as it is cheap and effective. Traditional cloning is characterized by the use of restriction enzymes which yield sticky ends. These sticky ends can be ligated to each other by a ligase. The ligated plasmid can subsequently be transformed. We used traditional cloning on and off as well as as a back-up plan if our Gibson Assemblies failed.
Figure A: Overview of the workflow of Traditional Cloning. Traditional Cloning uses restriction enzymes (the scissors) to cut DNA at specific places. Cutting the DNA yields distinct sticky ends which can be ligated together. By cutting both a to be inserted fragment as well as the vector, one can insert the fragment into a vector. As a result, one can obtain a new plasmid.
Figure A: Overview of the workflow of Traditional Cloning. Traditional Cloning uses restriction enzymes (the scissors) to cut DNA at specific places. Cutting the DNA yields distinct sticky ends which can be ligated together. By cutting both a to be inserted fragment as well as the vector, one can insert the fragment into a vector. As a result, one can obtain a new plasmid.
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.
General workflow of Gibson Assembly. The first step consists of linearizing the vector using either PCR or digestion by restriction enzymes. Next, the linearized vector and dsDNA fragments are introduced in a tube with the Gibson Assembly Master Mix, and incubated at 50°. The resulting mixture is transformed into competent cells and analyzed using colony PCR to select the correctly assembled vectors.
General workflow of Gibson Assembly. The first step consists of linearizing the vector using either PCR or digestion by restriction enzymes. Next, the linearized vector and dsDNA fragments are introduced in a tube with the Gibson Assembly Master Mix, and incubated at 50°. The resulting mixture is transformed into competent cells and analyzed using colony PCR to select the correctly assembled vectors.
- 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.
- NEBuilder HiFi Assembly - During our iGEM summer, we used the NEBuilder HiFi Assembly Kits. These kits contain a high-fidelity polymerase rather than a normal polymerase, limiting the occurence errors during the Gibson Assembly. This protocol contains the one-pot assembly method as well as transformation of the product into NEB 5-alpha cells