Lab protocols

• General lab safety and aseptic technique
• Making agar plates
• MSM minimal medium
• Growing E.coli
• Preparation of chemically-competent E.coli
• Heat shock transformation of chemically-competent E.coli
• Preparation of electrocompetent cells of Pseudomonas or E.coli, and electroporation
• Preparation of electrocompetent cells of Mycobacterium, and electroporation
• Resting cell experiments with bacteria
• Gas chromatography (Thermo Trace 1300)
• Making, running, staining agarose gels
• Polymerase chain reaction (PCR) and primer design
• Purification of PCRs or digests via spin column
• Purification of PCRs or digests from gel by spin column
• Genomic DNA extraction with CTAB
• Genomic DNA extraction with bead beater
• Plasmid miniprep – 5 ml – spin column
• Plasmid midiprep – 50 ml – spin column
• Plasmid midiprep – 50 ml – phenol-chloroform
• Quantitation and digestion of plasmid DNA 
• Golden Gate Assembly of DNA
• Cloning into plasmids – digestion, ligation, troubleshooting
• Screening recombinant clones (patch, PCR, digest)
• Validation of recombinant plasmids by Sanger Sequencing
• Protein analysis by SDS-PAGE

Plasmids

 

Addgene ID	Plasmid	Function	Publication
127674	pUS252	Expresses fuGFPb constitutively in E.coli. Modular template for cutting/amplifying fuGFP to place in other plasmids. fuGFPb absorbs UV, emits green.	A new open-source superfolding green fluorescent protein: Free-Use GFP (fuGFP) (unpublished)
84577	pMycoFos	Shuttle and expression vector. Replicates in E.coli and Mycobacterium. Gives acetamide-inducible gene expression in Mycobacterium. Copy number is controllable by arabinose in E.coli.	Construction and evaluation of pMycoFos, a fosmid shuttle vector for Mycobacterium spp. with inducible gene expression and copy number control.
84578	pUS116	Shuttle and expression vector. Replicates in E.coli and Mycobacterium. Gives acetamide-inducible gene expression in Mycobacterium.	Heterologous Expression of Mycobacterium Alkene Monooxygenases in Gram-Positive and Gram-Negative Bacterial Hosts
84579	pUS232	Shuttle and expression vector. Replicates in many Gram-negative bacteria. Has multiple cloning site with blue/white selection function. Tetracycline-inducible expression of cloned genes.	Coleman Lab plasmids (unpublished)
84580	pUS212	E.coli cloning and expression vector. Has cloning sites downstream of Tet promoter.	Coleman Lab plasmids (unpublished)
191831	pUS252b	Expresses fuGFPb constitutively in E.coli. Modular template for cutting/amplifying fuGFP to place in other plasmids. fuGFPb absorbs blue, emits green.	A new open-source superfolding green fluorescent protein: Free-Use GFP (fuGFP) (unpublished)
191832	pUS252L	Expresses fuBFP constitutively in E.coli. Modular template for cutting/amplifying fuGFP to place in other plasmids. fuBFP absorbs UV, emits blue.	A new open-source superfolding green fluorescent protein: Free-Use GFP (fuGFP) (unpublished)
191833	pUS252s	Expresses fuGFPs constitutively in E.coli. Modular template for cutting/amplifying fuGFP to place in other plasmids. fuGFPs absorbs UV, emits blue.	A new open-source superfolding green fluorescent protein: Free-Use GFP (fuGFP) (unpublished)
191834	pUS252y	Expresses fuYFP constitutively in E.coli. Modular template for cutting/amplifying fuGFP to place in other plasmids. fuYFP absorbs green, emits yellow	A new open-source superfolding green fluorescent protein: Free-Use GFP (fuGFP) (unpublished)
198322	pUS250	Provides cumate-inducible gene expression in diverse bacteria. Has large MCS, with blue/white screening via amilCP chromoprotein	pUS250: A new broad host range vector for inducible gene expression in diverse gram negative bacteria (unpublished)

External Links

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DATABASES

• PubMed
• NCBI nucleotide & NCBI protein
• Web of Science
• KEGG metabolic pathways
• Codon usage database
• IS finder insertion sequence database
• Tn Central transposon database
• Transposon registry & Integrall database
• iGEM Parts Registry

BIOINFORMATIC TOOLS

• BLAST
• Ribosome Binding Site Calculator
• Sequence Manipulation Suite
• Promoter predictor
• Terminator predictor
• RNA fold
• BioIcons free graphics

BIOSCIENCE COMPANIES

• IDT
• Twist
• NEB
• Merck-Sigma-Aldrich
• AGRF
• Plasmidsaurus
• SeqCenter