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Calcium Phosphate Transfection Method - http://www.flemingtonlab.com/Protocols/CalciumPhosphateTransf.pdf

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This calcium phosphate transfection method works best in cell lines that are 1) highly transformed and 2) adherent (Hela, U2OS, SAOS2, AdAH, NPC-KT and obtain from 20% to 100% transfection efficiency depending on the cell line). Works well for transient experiments but precautions should be used in the design and interpretation of experiments based on the discussion below. Also works very well for generating stable cell lines. This method is quite sensitive to the amount of input plasmid. - [Read Calcium Phosphate Transfection Method]

Calcium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3872

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol,is based on the venerable method of Graham and van der Eb (1973). The procedure is used chiefly to generate stable lines of cells carrying chromosomally integrated copies of the transfected DNA. - [Read Calcium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA]

Constructing and Expressing GFP Fusion Proteins - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4649

Category: Reporter Gene Assays: GFP Assay Protocols

The following protocol can be used for the development of stable cell lines expressing GFP fusion proteins. Although optimal transfection procedures (e.g., calcium phosphate, electroporation, or FuGENE 6 [Roche Applied Science]) vary depending on cell type, this general transfection procedure has been successful for stable transfection of HeLa, A-431, U2OS, BHK, and HT1080 cells. - [Read Constructing and Expressing GFP Fusion Proteins]

Culture Conditions for Various Stable Cell Lines - http://www.flemingtonlab.com/Protocols/CultureStableCellLines.pdf

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Culture conditions for various stable cell lines. - [Read Culture Conditions for Various Stable Cell Lines]

Culture of Human Prostatic Carcinoma Cell Lines - http://www.celldeath.de/apometh/rpmi.html

Category: Cell Biology and Cell Culture: Cell Line Culture Conditions

Culture of Human Prostatic Carcinoma Cell Lines, Growing and splitting the cells, Preparation of frozen stocks in liquid nitrogen, How to bring frozen cells back into culture, Concentrations of antibiotics for the selection of stable transfectants. LNCa - [Read Culture of Human Prostatic Carcinoma Cell Lines]

DNA Transfection Using Polybrene Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3875

Category: Transfection Protocols

Polybrene and DMSO can be used to achieve stable transformation of several types of cells by plasmid DNA. The yield of transformants is up to 15-fold greater with Polybrene than with calcium phosphate-DNA coprecipitation. However, there is no difference between the two methods in the efficiency of transformation of cells by high-molecular-weight DNA. - [Read DNA Transfection Using Polybrene Protocol]

Dose of G418 to be used for selection - http://invitrogen.custhelp.com/cgi-bin/invitrogen.cfg/php/enduser/std_adp.php?p_sid=rLTNWTng&p_lva=&p_faqid=80

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

What dose of Geneticin (G418) should I use for stable cell line selection? Invitrogen FAQs. - [Read Dose of G418 to be used for selection]

Establishment of Stable Transfectant of CHO Lec Cells Protocol - http://www.cbrinstitute.org/labs/springer/protocols/jun_CHOlec.html

Category: Transfection Protocols: Stable Transfection Protocols

CHO Lec3.2.8.1 cells have 4 independent mutations in the N and O glycosylation pathways. When cultured with alpha-glucosidase I inhibitor N-butyl-deoxynojirimycin, glycoproteins produced in CHO Lec3.2.8.1 cells are completely susceptible to Endo H digestion. Endo H cleaves chitobiose, leaving a single N-linked N-acetylglucosamine per site which is ideal for maintenance of protein solubility and special carb-protein interactions, such as between the first N-acetyl glucosamine residue and tryp. - [Read Establishment of Stable Transfectant of CHO Lec Cells Protocol]

Home-made Taq Polymerase Purification - http://www.nottingham.ac.uk/~mbzspd/methods/Home_made_Taq_polyme.html

Category: PCR Protocols

PCR polymerase costs can be high. If you are willing to work, you can produce bacteria containing the clone. It appears to produce lots of Taq and is quite stable. The proceedure takes 4 days start to (15 000 units of Taq) finish. The Taq also appears ver - [Read Home-made Taq Polymerase Purification]

Hybridization and Detection Using the HC ExpressArray Kit Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4122

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol describes the direct detection of RNA on DNA microarrays using Hybrid Capture (HC) technology and the HC ExpressArray Kit developed by Diagene. The kit uses a proprietary antibody that binds specifically to RNA:DNA hybrids and a second, fluorescently labeled, antibody that detects the primary antibody. Total RNA is applied directly to a glass-spotted DNA microarray, and stable RNA:DNA hybrids are visualized via a Cy3-labeled secondary antibody. - [Read Hybridization and Detection Using the HC ExpressArray Kit Protocol]

Peptide Handling and Storage - http://www.sigmaaldrich.com/Brands/Sigma_Genosys/Custom_Peptides/Key_Resources/Handling___Storage.html

Category: Peptide Protocols: Peptide Storage

In general, peptide solutions are stable for up to a week at 4Â°C. However, if the peptide sequence has inherent instability (Peptide Stability), it might be better to freeze the solution when not in use. Peptide solutions at pH>8 should also be frozen when not in use. Sigma Aldrich. - [Read Peptide Handling and Storage]

Protocol for Stable Lines - http://genetics.med.harvard.edu/~cepko/protocol/mike/S4.html

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

This protocol is optimized for erythroid cells, other cell types may require modifications. - [Read Protocol for Stable Lines]

Selection of Transfected Suspension Cells - http://www.musc.edu/BCMB/ceramide/protocols/0008.html

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

hygromycin, neomycin selection of stable cells in suspension. Suprya Jayadev / Dr.Hannun Dr.Obeid Lab, BCMB, South Carolina. - [Read Selection of Transfected Suspension Cells]

Tetracycline as Regulator of Inducible Gene Expression Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3986

Category: Transfection Protocols: Stable Transfection Protocols

Protocol uses an autoregulatory system in which the transcriptional trans-activator tTA drives its own expression and that of a target gene. The first stage of the procedure describes how to generate stable lines of NIH-3T3 cells that express either tTA alone or tTA and the tetracycline-regulated target gene. - [Read Tetracycline as Regulator of Inducible Gene Expression Protocol]

Transformation of Tetrahymena thermophila by Electroporation Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4502

Category: Tetrahymena Protozoa Protocols

Protocol describes a method for transformation of the Tetrahymena using electroporation. The vector is electroporated into cells after mating, where it is incorporated into the DNA of developing macronuclei. Because T. thermophila can be propagated indefinitely without conjugation, transformation of the macronucleus provides a way to obtain stable somatic transformants. DNA vectors transformed using this protocol include those containing drug-resistant versions of Tetrahymena genes. - [Read Transformation of Tetrahymena thermophila by Electroporation Protocol]

Transformation of Tetrahymena thermophila by Electroporation Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4502

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol describes a method for transformation of the Tetrahymenausing electroporation. The vector is electroporated into cellsafter mating, where it is incorporated into the DNA of developingmacronuclei. Because T. thermophila can be propagated indefinitelywithout conjugation, transformation of the macronucleus providesa way to obtain stable somatic transformants. - [Read Transformation of Tetrahymena thermophila by Electroporation Protocol]

Articles (1-3 of 3)

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol

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.

Picking Transfected ES Cell Clones Protocol

This protocol a protocol on how to generate transfected embryonic stem (ES) cell clones. The previous protocol in this series is the Protocol for Electroporation of ES cells. The next protocol in the series is the Protocol on Disaggregation, Expansion, and Freezing of Transfected ES Clones.