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In this video, Dr. Sean Taylor, Field Applications Specialist, Bio-Rad Laboratories, demonstrates how sample quality and reference gene selection effect data analysis and interpretation in real-time quantitative PCR (qPCR) experiments. The presentation is in accordance with the previously published MIQE guidelines.
For enhanced viewing, click on the full-screen mode button on the bottom right hand corner of the video.
Can life begin ex nihilo (from nothing)? That is the question that scientists and theologians have been asking for centuries. A new study out of the University of Penn State University suggests that the success of early life forms may have indeed begun from from non-living matter.
Christine Keating, an associate professor of chemistry at Penn State University, and Meghan Andes-Koback, a graduate student in the Penn State Department of Chemistry, generated simple, non-living model “cells” with which they established that asymmetric division — the process by which a cell splits to become two distinct daughter cells — is possible even in the absence of complex cellular components, such as genes.
The new modeling techniques seems to suggests that simple chemical and physical interactions within cells — such as self-assembly, phase separation, and partitioning — can result in seemingly complex behaviors – like asymmetric division — even when no additional cellular machinery is present.
Furthermore, the fact that the rudimentary process of cell division, (excluding cell differentiation and a myriad of cell functions), can occur in the absence of genetic material and other cellular machinery, provides evidence that the mysterious process of abiogenesis — the formation of life from non-living matter — is indeed a scientific possibility.
To read more click New Technique Sheds Light on the Mysterious Process of Cell Division
Most people who have run polyacrylamide gels have at one point or another gotten confused with the gel’s orientation. This is especially true following protein transfer from the gel to a membrane for the purpose of western blotting or other downstream processing. This can be extremely frustrating and may even jeopardize your entire experiment if you are unable to tell the right side of the gel from the left or your control samples from your treatment group.
Several methods have been created to help bench scientists avoid this problem. These include the use of multicolored protein ladders and marking a predefined corner of your membrane once the protein has been transfered (I cut the bottom left corner of the membrane).
Today, Arefeh Seyedarabiclose from the Department of Structural and Molecular Biology, University College London published a new and very basic method for labeling polyacrylamide gels on the Nature Protocol Exchange website. Essentially, Arefeh suggests labeling the bottom inside corner of the long glass plate (facing the gel) with permanent marker). During electrophoresis the label will transfer from the plate to the gel, thereby permanently labeling your gel. Simple and brilliant!
To see the full method and associated figures click on A method for labeling polyacrylamide gels.
If you would like to share other great molecular biology methods, please post a comment or send us an email using the email button at the top of the page.
Reference:
Arefeh Seyedarabi, A method for labeling polyacrylamide gels, Protocol Exchange (2011) doi:10.1038/protex.2011.222, Published online 24 March 2011
Most of my graduate work focused on transcriptional regulation of a vasoregulatory gene and all the nitty-gritty work that goes along with these types of molecular protocols. As such, I am always on the lookout for techniques that improve upon current transcriptional regulation protocols especially if they show a propensity for doing the job either faster or better than is currently done.
In a study published today in Nature, L. Stirling Churchman et al from the University of California, San Fransisco reveal a technique that utilizes fast DNA-sequencing technology and advanced computer technology to examine transcriptional regulation with unprecedented resolution in real-time and in-vivo.
The native elongating transcript sequencing protocol, (NET-seq), is based on deep sequencing of 3′ ends of nascent transcripts associated with RNA polymerase, to monitor transcription at nucleotide resolution.
Until quite recently, many scientists thought that less than 5 percent of the human genome was actually transcribed into RNA and therefore used in the cell’s function, Churchman said. Recent advances in the field have revealed a tremendous complexity in that process, with new understanding that the majority of DNA is transcribed. Much of the product is still considered “junk RNA” – simply a byproduct of the process.
“Now, the question is not, ‘Why is that DNA there?’ but, ‘Why is that RNA there?’” said Churchman, a physicist and post-doctoral scholar at UCSF. “It could be junk RNA, but we don’t know.”
In the video below you can see how DNA is tightly wound around histones forming a nucleosome complex that is then packaged into the cell. Binding of transcriptional proteins to target DNA is not only contingent upon the DNA sequence but also depends upon the accessibility of the DNA template which depends upon how it is wrapped around the histone. As you can see in the video, some DNA remains accessible by virtue of the fact that it faces “outwards” while other portions of the DNA are harder for the transcriptional proteins to access.
In the present study, scientists were able to observe for the first time that polymerase comes in direct contact with the histone proteins during the transcription process, while also seeing how the nucleosomes acted as a speed bump for the polymerase enzyme as it moved along the genome transcribing DNA into RNA. In addition, the research showed that the organization of histone marks controlled whether “junk RNA” was produced from a given region of DNA.
Visit the UCSF site to read more.
Reference:
L. Stirling Churchman, & Jonathan S. Weissman (2011). Nascent transcript sequencing visualizes transcription at nucleotide resolution Nature, 469, 368-373 : doi:10.1038/nature09652
High resolution melt (HRM) analysis is a relatively new technique used in detecting small variations in DNA sequences between varying populations. Important applications of HRM include SNP analysis, genotyping and methylation analysis.
In the following 20 minute tutorial presented by Sean Taylor, Field Application Specialist, Bio-Rad Laboratories, you will learn the basics of high resolution melt analysis and how to practically use it in your research. The video contains information on:
For more information on HRM see A Practical Guide to High Resolution Melt Analysis Genotyping
