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.
Transformations
Transformation refers to the act of introducing new plasmid DNA into bacteria. The plasmids can be introduced in bacteria in numerous ways. Perhaps the most well-known methods are electroporation and heat shocking of competent cells. In our project, we used competent cells exclusively. These cells have been chemically modified to transform efficiently. Competent cells can be directly ordered from a wide range of life sciences companies. We used BL21DE3, NovaBlue and NEB 5-alpha cells exclusively.
Figure C: Transformation is a term used for the introduction of new plasmids into bacteria. Transformation can have place in numerous ways. Often, the term is used intechangeably with transfection. This latter term is, however, reserved for the introduction of plasmid DNA into eukaryotic cells.
Figure C: Transformation is a term used for the introduction of new plasmids into bacteria. Transformation can have place in numerous ways. Often, the term is used intechangeably with transfection. This latter term is, however, reserved for the introduction of plasmid DNA into eukaryotic cells.
Gibson Assembly
Gibson Assembly is a one-pot assembly method. Gibson Assembly requires a linearized vector as well as dsDNA fragments. Linearization of the vector can be realized by the use of restriction enzymes or through PCR. The dsDNA fragments can be obtained through PCR or they can be ordered directly from a manufacturer. In our project, we linearized the vector through PCR and ordered dsDNA fragments directly from IDT.
Figure C: Figure 1 - 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.
Figure C: Figure 1 - 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