To 2mls of mid-log culture or 1ml of freshly saturated culture add 1ml(or an equal volume) of glycerol solution, mix gently, then freeze rapidly in liquid nitrogen or on dry ice. Store at -20°C and - 70°C. - [Read Bacterial Glycerol Stocks Protocol]
DNA Affinity Chromatography, DNA affinity chromatography can be a low-tech method using gravity flow at 4°C, a disposable chromatography column, and DNA affinity resin prepared in the laboratory (see Preparation of a DNA Affinity Column). Include 10-20% glycerol and 0.025-0.1% NP-40 in the column buffers to suppress losses due to nonspecific adsorption of protein to surfaces. Load the protein in a buffer that is compatible with binding of the protein to its target site. Keith Brocklehurst et al - [Read DNA Affinity Chromatography Using Gravity Flow - Subscription Required]
Plasmid (pUC series) containing genomic DNA fragments are maintained in E. coli strain DH5aTM. The E. coli cultures are routinely cultured at 37 C on Luria-Bertani (LB) agar on or in LB broth containing Ampicillin (30 µg/ml) or Carbenicillin (50 µg/ml broth, 100 µg/ml agar). E. coli strains are usually preserved in stab agar or glycerol for mid-term storage and lyophilized for long-term storage. - [Read Maintenance of Probes in Bacteria Including Escherichia coli Protocol]
Electrocompetent bacteria are prepared by growing cultures tomid-log phase, washing the bacteria extensively at low temperature,and then resuspending them in a solution of low ionic strengthcontaining glycerol. DNA is introduced during exposure of thebacteria to a short high-voltage electrical discharge.
Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.
In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.
A protocol for the selection of Phage Antibodies using Immobilized Antigen. This method describes the selection of antibodies from bacteriophage antibody libraries that recognize a specific antigen. The phage display library of antibody-displaying phage particles is exposed to antigen attached to a solid substrate (Nunc Immuno™ tubes). The phage particles with affinity for antigen bind to the immobilized antigen and are selected from the library of phage expressing antibodies.
Histone H1 Kinase Activity Assay Protocol. This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography.