depending Protocols

Category: PCR Protocols: cDNA Synthesis Protocols

An oligodeoxynucleotide primer hybridized to mRNA is extended by an RNA-dependent DNA polymerase to create a cDNA copy that can be amplified by PCR.  Depending on the purpose of the experiment, the primer for first-strand cDNA synthesis can be specifically designed to hybridize to a particular target gene, or a general primer such as oligo(dT) can be used to prime cDNA synthesis from essentially all mammalian mRNAs - [Read Amplification of cDNA Generated by Reverse Transcription of mRNA Protocol]

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]

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]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Polyacrylamide Gels Protocols

The "crush and soak" method, which works best for DNAs <1 kb in size, can be used to recover both singleand double-stranded DNAs from polyacrylamide gels.  The yield of eluted DNA varies from <30% to >90% depending on the size of the DNA fragment. - [Read Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method Protocol]

Category: Stem Cell Protocols

This protocol describes passage of ES cells. They should be split at 1:3 to 1:7 every 2-3 days depending on their growth rate when they reach 70% confluency. They should never be allowed to grow past 90% confluency, but rather they should form tightly packed colonies not touching each other. - [Read Passage of Embryonic Stem (ES) Cells Protocol]

Category: Microinjection Protocols: Microinjection Equipment

This protocol contains methods for pulling microinjection needles using two different models of pipette pullers. The advantage of pulling needles in the laboratory is that a variety of different needle types can be pulled, depending on the samples and cells being injected. An added advantage is cost; once a pipette puller has been purchased, boxes of glass capillaries are inexpensive compared to premade microinjection needles. - [Read Preparation (Pulling) of Needles for Gene Delivery by Microinjection Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for preparation of KC nuclear extract for in vitro splicing. Protocol makes 3.4 ml of extract for every 4 liter of cells (depending on initial cell concentration). Protocol includes: Procedure, Solutions, BioReagents and Chemicals and protocol hints. - [Read Preparation of KC Nuclear Extract for In Vitro Splicing]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

The methodology and buffers utilized for processing microdissected samples for protein-based studies is variable depending on the downstream molecular analysis method. - [Read Processing of Microdissected Tissue for Molecular Analysis Proteomics-based Studies]