Team:Leicester/labwork/methods
Methods
The methodology to make biobricks are similar to the interlab measurement study which includes:
- Transformation using NEB Chemically Competent Cells (to obtain a high transformation efficiency) using the iGEM protocol which can be found at: Transformation Protocol
- Liquid Culture- inoculating a colony overnight in a universal tube containing 2ml LB with 35ng/µl Chloramphenicol.
- Miniprep- using the (SIGMA) Miniprep kit following the protocol to enable the isolation of the transformed plasmid DNA from the bacteria.
- Nanodrop- Using elution buffer as a blank to identify the concentration of the minipreped DNA.
- Digestion- using the 3A assembly protocol if parts need to become digested. For digesting PCR products with the iGEM prefix and suffix only EcoR1-H1 and Pst1 were used. This was used to create sticky ends for ligating parts together or into a C3 backbone.
- Ligation- Using the 3A assembly protocol for ligation to ligate parts together or into the pSB1C3 backbone. The protocol for the 3A assembly is found here: 3A Protocol
- PCR- using primers designed by the team to PCR amplify out NadD, NadE and PncB from E.coli genomic DNA, as well as removing the Pst1 site from PncB through the use of internal primers through the Phusion PCR protocol.
- Gel Extraction- using the (SIGMA) Gel Extraction kit protocol to extract and purify PCR products that have been run in an 1.5% Agarose Gel.
- Gel Electrophoresis- making and using 1%1.5% or 3% Agarose Gels for analytical purposes to confirm digestion and or ligation has worked, as well as, confirming the PCR products are the right size and whether the PncB site has had the Pst1 restriction site removed.