Protocols¶

In general, the sequence of experimental steps to obtain a certain result is specified in so called protocols. It is central to the scientific method that protocols are shared among researchers so that experiments can be reproduced. Protocols can be found in Methods sections of papers, although often steps that are assumed to be known, are missing - thus making it difficult to get started.

In greater detail, protocols are shared by:

dedicated journals like Nature methods
websites like protocols.io
companies that sell consumables for protocols (for example NEB)

A nice start into basic protocols and procedures like pipetting and centrifugation can be found at www.addgene.org/protocols/. We also found their videos really helpful.

We present a particularly important technique in greater details: cloning of a plasmid.

Common Protocols¶

Here's a quick reference of protocols you may often need when engineering bacteria.

Stock and Grow Bacteria¶

Protocol	Purpose	Link
Creating a glycerol stock	Long-term storage of bacterial strains/plasmids at −80°C	https://www.addgene.org/protocols/create-glycerol-stock/
Inoculate bacterial culture	Start an overnight E. coli culture from a colony or glycerol stock	https://www.addgene.org/protocols/inoculate-bacterial-culture/

Extract DNA¶

Protocol	Purpose	Link
Plasmid miniprep	Isolate plasmid DNA from E. coli	https://www.addgene.org/protocols/purify-plasmid-dna/ and https://www.neb.com/en/products/t1110-monarch-spin-plasmid-miniprep-kit
PCR amplification	Amplify DNA fragments for cloning	https://www.addgene.org/protocols/pcr/
Gel extraction	Purify DNA fragments from agarose gels	https://www.neb.com/en/products/t1120-monarch-spin-dna-gel-extraction-kit

Cloning¶

Protocol	Purpose	Link
Restriction digest / linearization	Cut or linearize plasmid DNA with restriction enzymes	https://www.addgene.org/protocols/restriction-digest/
Gibson Assembly	Seamless assembly of overlapping DNA fragments	https://www.addgene.org/protocols/gibson-assembly/ and https://www.snapgene.com/guides/gibson-assembly
Bacterial transformation	Introduce plasmid DNA into competent E. coli	https://www.addgene.org/protocols/bacterial-transformation/