To ensure that the experiments could be carried out efficiently and safely and reproduced accordingly, we introduced the following standard operation procedures (SOPs). This was necessary since we were working with 13 people in the laboratory. These SOPs contain detailed information on the chemicals and devices needed, bullet point manuals, and information on risk factors and proper disposal. In the beginning, there was a lot of questioning about how to perform the experiments. We were able to reduce this through the SOPs, as the standardization helped to enable each member of our laboratory team to work independently, reproducibly and - most importantly - safely.
Buffers and Solutions
These protocols serve as a solid basis for daily work in the laboratory with gel electrophoreses, cloning and DNA synthesis.
Agarose Gel Electrophoresis
Since agarose gel electrophoresis was our most valuable and used analysis tool, the folllowing protocols for gel and sample preparation along with performing the electrophoresis were often used in the laboratory.
PAA Gel Electrophoresis
These protocols ensured the correct preparation and performance of native and denaturing polyacrylamide gel electrophoreses. Staining of the gels with silver solution and SYBR Gold was also documented.
TdT Tailing Reaction
After initial testing with the TdT, a standard TdT reaction was established from which reactions could be varied to characterize the TdT even further. It was also documented that the TdT can be inactivated with heat and EDTA.
The use of some devices in the laboratory was not trivial. Thus, we wrote protocols to enable simple and safe operation of the devices without having to ask a laboratory supervisor.
Preparation for Sanger sequencing includes casting agar plates, digestion of a vector, cloning of a vector, and a colony polymerase chain reaction. The protocols serve to ensure that everyone in the laboratory team was able to perform these preparations.
To properly perform immobilization with the magnetic beads, we established protocols on how the biotin-streptavidin bond is formed as well as separated and the cyclic synthesis can be performed in the immobilized state.