Protocol applies EFs to cells in vitro but has been modified and to use electrotactic chambers to accommodate cells growing in planar culture or in three-dimensional (3D) gels, en bloc tissue cultures in 3D and possible small embryos, such as that from frog and zebra fish. The EF is applied to the cells or tissues cultured in a customer designed electrotactic chamber via agar salt bridges, Steinberg’s solution and Ag/AgCl electrodes. - [Read Application of Direct Current Electric Fields to Cells and Tissues in vitro]
Arabidopsis transformation and selection on kanamycin agar, Preparation of plants for transformation, Agrobacterium, Dipping, Post-dip care, Agar plates, Tips. Adaptation of the short protocol described bySteve Clough and Andrew Bent, University of Illinois at Urbana-Champaign. - [Read Arabidopsis transformation with pictures]
Most manipulations with M13, including preparations of viral stocks and isolation of single- and double-stranded DNAs, begin with small-scale liquid cultures that are infected with an M13 plaque, picked from an agar plate. - [Read Growing Bacteriophage M13 in Liquid Culture Protocol]
To image early cleavages and chromatin dynamics, it is convenient to use histone H2B fused to GFP or lamin::GFP. Time-lapse movies can be obtained using conventional confocal microscope systems and their included software. Early embryos dissected from transgenic hermaphrodites are placed with egg salts on agar pads. - [Read Live Imaging of Caenorhabditis elegans: Examples]
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]
Media for Culture of Mammalian Cells Protocol- https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E662B6F09C1BB8FBC47FC2A7E61A8A3&objectid=66739B890A55AC13354418E67F28E693
The culture medium is an essential component of the in vitro environment and must be selected or designed with care. This protocol provides guidelines for design of serum-containing and serum-free media, selective and specialty media, and media for growth under special conditions such as soft-agar growth. - [Read Media for Culture of Mammalian Cells Protocol]
Protocols for Neurospora methods. Includes: Standard strains; Crosses; Minimal medium; Color coding of media; Agar substrate for manipulation and isolation; Stock solutions of supplements; Mating-type tests; Preservation of stocks by silica gel; Cleaning of glassware; Control of mites. - [Read Neurospora Methods Protocols]
To image early cleavages and chromatin dynamics, it is convenient to use histone H2B fused to GFP or lamin::GFP. Time-lapse movies can be obtained using conventional confocal microscope systems and their included software. Early embryos dissected from transgenic hermaphrodites are placed with egg salts on agar pads. Chromatin dynamics can be followed easily, and wild-type embryonic cells can be compared with mutants or RNAi-treated embryos. - [Read Protocol Live Imaging of Caenorhabditis Elegans]
Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the filters are processed for hybridization to an appropriate radiolabeled probe while the original (master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their original positions. - [Read Screening Bacterial Colonies by Hybridization: Intermediate Numbers Protocol]
Protocol used to screen a small number of bacterial colonies (<200) that are dispersed over several agar plates and are to be screened by hybridization to the same radiolabeled probe. The colonies are gridded onto a master plate and onto a nitrocellulose or nylon filter laid on the surface of a second agar plate. - [Read Screening Bacterial Colonies by Hybridization: Small Numbers Protocol]
A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.
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.
3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol. This protocol contains the steps for 3' end rapid amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly-A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.
This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.