inserts Protocols

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Manual measurement and manipulation of the cell surface requires access to the cells, usually in an open chamber. Temperature-controlled chambers or stage inserts are preferred for maintaining physiological activity during the experiment. For example, heated culture dishes with coverslip glass bottoms (Bioptechs) permit high-resolution fluorescence microscopy of living cells during force application. - [Read Chambers for Examination of Live Cells under Mechanical Stress Protocol]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

Shotgun sequencing of a large segment of DNA involves random fragmentation of the target region into smaller segments that are subsequently cloned into a bacteriophage M13 vector. The goal is to create a library of overlapping clones that provide at least fivefold coverage over the entire length of the target fragment. - [Read Generation of a Library of Randomly Overlapping DNA Inserts Protocol]

Category: PCR Protocols: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]

Category: DNA Microarray Protocols

This protocol describes PCR amplification of eukaryotic cDNA plasmid inserts, gel electrophoresis, purification, and storage of PCR products. Hasseman. TIGR Microarray Protocols. - [Read MICROARRAY PCR, PURIFICATION, AND STORAGE]

Category: Cloning Protocols: Vector Protocols

To minimize self-ligated vector in your transformation, treat your linearized vector with a phosphatase to remove the 5' phosphates necessary for ligation. This should improve the percentage of colonies with inserts. - [Read Phosphatase Treatment of Linearized Vector Protocol]

Category: Fungi Protocols: Neurospora Protocols

Protocol guide for the N. crassa yeast artificial chromosome library. Includes: Chromosome Walking; Hybridization screening of the YAC library; YAC restriction mapping and contig building; Preparation of chromosomal DNA plugs of YAC clones; Partial restriction enzyme digestion of YAC DNA plugs; Using CHEF gel analysis to resolve YAC clones; Southern Hybridization; Isolation of terminal restriction fragments from cloned DNA inserts in YAC clones; etc. - [Read Protocol Guide for the N. crassa Yeast Artificial Chromosome Library]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

A vector that lacks inserts can significantly contaminate a DNA library. This contamination can occur either from self-ligation of single-cut vector or from uncut circular starting plasmid. - [Read Synthesis of DNA Libraries: Use of PCR to Analyze Ligation Substrates and Products]