This is a validated protocol
Representative data for this protocol can be found here.
This protocol creates a 1800µL bacterial culture from either another bacterial culture or a glycerol stock. Once the culture has reached sufficient cell density the culture is lysed and the plasmids within the cells are extracted and purified. Purified plasmids are then returned to you inventory for down stream applications.
An aliquot of a bacterial culture is required. Samples should be either in glycerol stock or bacterial broth. 10µL of the sample will be used in the protocol, so please make sure you select a sample with at least 10µL in addition to the dead volume of that container.
To add more bacterial aliquots click the 'Add Bacterial sample(s) from which to purify DNA' link.
PURIFIED DNA LABEL
For each bacterial sample you select, you are also required to create a label for the purified DNA that is produced from the aliquot.
ANTIBIOTIC SELECTION MEDIUM
Prior to plasmid extraction & purification, bacteria will be incubated for 16h at 37C in this medium, to amplify cell density.
For each aliquot that you select, this protocol will produce a corresponding aliquot of purified DNA that will automatically be added to your inventory and will be labeled as specified when launching the protocol. This aliquot will be stored at -20C.
For bacterial sample inputs, take an OD600nm reading of each source aliquot using our Plate Reading quick instruction. The read should be above the background value of 0.05, meaning there's bacteria present. If you are using an aliquot that was the output of the Transformation, Spread & Pick protocol, refer back to the results of this protocol for the OD600nm readings.
When using glycerol stocks, ensure that bacteria are present before creating the stock. Repeated freeze-thaw cycles will negatively affect the glycerol stock, so we recommend storing multiple smaller aliquots at -80.
This protocol uses the ISOLATE II Plasmid Mini Kit from Bioline, which has a maximum plasmid size specification of <15kb. Isolating plasmids above 15kb is not recommended.
Check out the forum if you have more questions.
For further advice on your protocol please ask questions to the community at https://forum.transcriptic.com
Updated 11 months ago