Team:Oxford/Protocols
Protocols
Under constructionCloning
Overview Cloning is the process of enclosing our Parts into plasmids, which are then transformed inside our E. coli bacteria. For each part, there is a structured pathway, which is summarised below:Day 1: PCR > gel > gel extract > digest > ligation overnight
Day 2: transformation
Day 3: pick colonies
Day 4: miniprep > diagnostic gel > send for sequencing
We perform the Polymerase Chain Reaction (PCR) to amplify the Part by many orders of magnitude. Next, we run the PCR product on an agarose gel, excise out the desired gel band, and extract the DNA from the gel. Then, we digest the Part with specific restriction enzymes; the same enzymes are used to digest the vector. To send to iGEM, all parts must be put into the shipping vector – pSB1C3 – and for parts which lack a promoter, these must additionally be put into an expression vector – pBAD – which is under control by a constitutive arabinose promoter. After overnight ligation, we transform our plasmids into competent cells by the heat-shock method. We spread our transformed cells onto antibiotic plates, in order to select for successful ligations and transformations (as the plasmid has an antibiotic resistance gene). On day 3, we pick colonies which appear on the plate; each derives from a single parental line. We leave these colonies to grow overnight in culture. On day 4 of the procedure, we purify the DNA out of the bacteria. We take an aliquot of this miniprepped sample, digest again with the same restriction enzymes and run this on a gel. If we see the desired band size (part size) and the vector band size on the gel, then we send an aliquot of the miniprepped sample for sequencing, to check that the Part is as expected. Assuming this is the case, the miniprepped sample is then transformed into the E. coli strain MG1655 for characterisation and testing.
