Universidad Autonoma de la Ciudad de Mexico

Electrophoresis through agarose or polyacrylamide gels is used to separate, analyze, identify, and purify DNA fragments. The technique is simple, rapid to perform, and capable of resolving fragments of DNA that cannot be separated adequately by other procedures, such as density gradient centrifugation

This protocol describes how to use northern hybridization to detect 15- to 150-nt small RNAs.

In this protocol, cells transfected with an Escherichia coli transposon chloramphenicol acetyltransferase (CAT) reporter plasmid are lysed by repeated cycles of freezing and thawing and cellular debris is removed by centrifugation.

Adenylation status has an important role in the regulation of mRNA metabolism: mRNAs are deadenylated before degradation, microRNAs (miRNAs) can cause deadenylation, and the poly(A) length of certain mRNAs is regulated during development. This protocol describes methods that can be used to measure the poly(A) tail length of specific mRNAs. These include, in the order of increasing sensitivity, (1) northern blotting of intact and experimentally deadenylated mRNAs and (2) northern blotting of inta...

The RNase protection assay is a sensitive method for transcription start-site localization. It begins withan RNA probe that is uniformly labeled by incorporation of one [α-32P]NTP, usually [α-32P]UTP. The RNA probe is synthesized by bacteriophage RNA polymerase (SP6, T7, or T3), which initiates transcription from specific phage promoters that have been engineered into a number of common plasmid vectors. The plasmid template contains a genomic DNA fragment spanning the region thought to contain...

Overlap extension polymerase chain reaction (PCR) mutagenesis can be used for the generation of a specific point mutation, insertion, or deletion within a particular DNA sequence of interest. It requires relatively little preparation compared with other mutagenesis methods and does not require the use of restriction enzymes.

“Salt-loving” microorganisms include a great variety of heterotrophic and methanogenic archaea; photosynthetic, lithotrophic and heterotrophic bacteria; and some photosynthetic and heterotrophic eukaryotes (such as algae, protozoa and certain fungi).