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Caprara M.,CSHL Press
Cold Spring Harbor Protocols | Year: 2013

Information about the secondary structure of RNA is often useful when assessing the potential for certain RNAs to interact with proteins or when determining whether RNAs that are dissimilar in sequence can form the same structure. In this protocol we discuss chemical methods for RNA structure determination. These methods rely on the fact that certain reagents interact with RNA bases when they are single stranded, but do not react when the bases are involved in Watson-Crick base pairs. For example, dimethylsulfate (DMS) methylates the N1 position of adenosine, the N7 position of guanine, and the N3 position of cytosine only when these bases are in single-strand regions. Modifications of adenosine and cytosine create blocks to reverse transcriptase; accordingly, these modifications are detected as stops to primer extension. Modification of guanine does not create reverse transcriptase stops, but these modifications can be detected by cleavage of the modified RNA after borohydride reduction and aniline cleavage. Because DMS and other chemical reagents modify only single-stranded RNA, double-stranded regions are inferred by the lack of modification. © 2013 Cold Spring Harbor Laboratory Press.

West M.J.,CSHL Press
Cold Spring Harbor Protocols | Year: 2013

Stereology involves sampling structural features in sections of tissue with geometrical probes. This article discusses some practical issues that must be dealt with when getting started in stereology, including tissue preparation methods and determining how many tissue sections and probes are needed to make a stereological estimate. © 2013 Cold Spring Harbor Laboratory Press.

Rio D.C.,CSHL Press
Cold Spring Harbor Protocols | Year: 2013

For large-scale transcription reactions or for cost savings, a laboratory may want to prepare its own recombinant T7-, SP6-, or T3-phage RNA polymerases. It is convenient to perform this preparation every 2-3 years and have a consistent and reliable source of phage RNA polymerase for many in vitro transcription reactions. In the protocol presented here, the recombinant plasmid expressing T7 RNA polymerase (RNAP) as a his6-tagged molecule is under an isopropyl β-D-1-thio-galactopyranoside (IPTG)-inducible promoter. The bacteria are lysed by sonication, the his6-tagged protein in the bacterial lysate is purified by binding to Ni-NTA agarose, and the resin is then extensively washed and eluted with imidazole. The purified enzyme is dialyzed against a glycerol-containing storage buffer and can then be stored for months or years at -20°C. © 2013 Cold Spring Harbor Laboratory Press.

Hagendorf N.,CSHL Press | Conzelmann K.-K.,CSHL Press
Cold Spring Harbor Protocols | Year: 2015

G-deleted fluorescent rabies virus (RV) pseudotyped with RV G proteins, SAD ΔG eGFP (RV CVS-G), can be used as single-round vectors for efficient retrograde labeling of neurons. For these experiments, as well as for monosynaptic tracing, which involves pseudotyping in situ, the use of the CVS strain G is recommended because of its high tropism for neurons. Pseudotype virus stocks generated by transfection of pCAGGS-G (or in MG139-on cells) contain the G protein of the vaccine strain SAD L16, which is broader in its tropism, and infects astrocytes, glia, and oligodendrocytes. We also describe a procedure for pseudotyping with ASLV Env A, which uses a cell-line expressing a version of the EnvA protein that is incorporated efficiently into the RV envelope (EnvARGRGct). © 2015 Cold Spring Harbor Laboratory Press.

Ohki K.,CSHL Press | Reid R.C.,CSHL Press
Cold Spring Harbor Protocols | Year: 2014

Two-photon imaging of calcium-sensitive dyes in vivo has become a common tool used by neuroscientists, largely because of the development of bolus loading techniques, which can label every neuron in a local circuit with calcium-sensitive dye. Like multielectrode recordings, two-photon imaging paired with bolus loading provides a method for monitoring many neurons at once, but, in addition, it provides a means for determining the precise location of every neuron. Thus, it is an ideal method for studying the fine-scale functional architecture of the cortex and guiding the experimenter to individual neurons that can be targeted for further anatomical study. Two-photon calcium imaging enables study of the fine structure of functional maps in the visual cortex in cats and rodents. In mice, it can allow the characterization of specific cell types when paired with transgenic or retrograde labeling. © 2014 Cold Spring Harbor Laboratory Press.

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