Protocols

Safety

See our Safety Page for Associated risk assessments for the protocols below and MSDS sheets for the reagents we have used.

Methods

Construct production

• Gel Electrophoresis A technique for separating DNA strands of different lengths.
• Gel Extraction of DNA A technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis.
• Gibson Assembly A technique for ligating multiple DNA fragments in one step, compatible with standard assembly.
• PCR using a high temperature DNA polymerase A method for amplifying a section of DNA.
• Making a Biobrick A final step to put your construct into the standard biobrick format to be submitted to the registry.

Transformation protocols

• Chemically competent cells generation A technique to produce e.coli cellsreceptive to chemical transformation.
• Electocompetent cells generation A technique to produce e.coli cells receptive to transformation by electroporation
• Electroporation A method for transforming appropriately competent cells with plasmid DNA using electricity.
• Glycerol stocks A technique for long term storage of cells at -80 degrees without losing cell vitality.
• Transformation of Bacillus subtilis A technique used to introduce foreign DNA into competent Bacillus cells.
• Transformation of Escherichia coli A method for transforming competent E.coli with DNA

Construct verification

• Colony PCR PCR with cells as a template. Useful for checking the length of an insert in an introduced plasmid.
• Restriction Enzyme Digest A method for creating a restriction map of a plasmid.
• MiniPrep - DNA extraction A method used to extract DNA from bacterial cells.

Ribosense testing

• β-galactosidase assay Assay to qualitatively measure the amount of the enzyme β-galactosidase being produced by a population of cells. Useful as a reporter system.
• Miller assay Assay to quantify the amount of the enzyme β-galactosidase being produced by a population of cells. Useful as a quantitative reporter system.
• Production of magnesium free cells. Technique to produce cells that contain little or no magnesium, to control for this variable in tests involving the magnesium riboswitch.

Ratiometrica testing

• IPTG induction A technique for inducing the pSPANK promoter in E.coli cells.

Sporulation and Germination

• Imaging spores A method for seeing vegetative cells and bacillus spores under the microscope.
• Producing high yields of spores A method for obtaining high yields of spores from B. Subtilis

Miscellaneous

• BioBricks Resources for manipulation of BioBricks and information regarding the distribution.
• LB Agar Plates preparation A method used to prepare agar plate to culture common bacteria.

Recipes

• M9 minimal medium A recipe for making M9 minimal medium for use when autofluorescence must be avoided.
• Medium B A recipe for making medium B.
• CCMB80 for chemically competent cells generation A recipe for making CCMB80
• TAE buffer for gel electrophoresis A recipe for making 10xTAE buffer
• SOB growth medium A recipe for making SOB growth medium
• CDSM A Chemically Defined Media to induce sporulation in B. Subtilis
• Germination Media A Chemically Defined Media to induce Germination in B. Subtilis

Lab supplies

• [http://www.bioc.cam.ac.uk/stores/ BioPath Stores]
• [http://www.plantsci.cam.ac.uk/camonly/stores/stock.xls Plant Department Stores Catalogue xls (Updated 2nd July 2012)]

Other resources

File:Concentration calculator for lazy scientists.xls

[http://meta.wikimedia.org/wiki/Help:Contents Media wiki editing]

[http://blast.ncbi.nlm.nih.gov/Blast.cgi BLAST at NCBI]

[http://www.finnzymes.fi/tm_determination.html finnzymes Tm calculator]

[http://mfold.rna.albany.edu/?q=mfold mfold], DNA or RNA folding forms on the left hand side