This is the second in a series of four blogs about Quantitation for NGS is written by guest blogger Adam Blatter, Product Specialist in Integrated Solutions at Promega. Perhaps the most ubiquitous quantitation method is UV-spectrophotometry (also called absorbance spectroscopy). This technique takes advantage of the Beer-Lambert Law: an observation that many compounds absorb UV-Visible […]
Robert Hooke first coined the term “cell” after observing plant cell walls through a light microscope—little empty chambers, fixed in time and space. However, cells are anything but fixed. Cells are dynamic: continually responding to a shifting context of time, environment, and signals from within and without. Interactions between the macromolecules within cells, including proteins, are […]
In 1982, picked up because of its homology to chicken virus genes that could transform cells, MYC became one of the first human genes identified that could drive cellular transformation (1,2). Since that time countless laboratories have prodded and poked the human MYC gene, the MYC protein, their homologs in other animal models, and their transforming […]
While T-vector cloning is commonly used for PCR-amplified inserts, restriction enzymes still have their uses. For example, you can ensure directional cloning if you digest a vector with the same two enzymes like BamHI and EcoRI that are used to digest your insert. This way, the insert can only be cloned in one direction. However, […]
Back in the dark ages, when I was a graduate student, my idea of “automated” plasmid DNA extraction involved performing home-brew, “toothpick preps” in “strip tubes” or , if I was really feeling ambitious, a 96-well plate. I would get just enough DNA to check for the presence of an insert, but the quality of […]
In the CheckMate™ Mammalian Two-Hybrid System, the pBIND Vector contains the yeast GAL4 DNA-binding domain upstream of a multiple cloning region, and the pACT Vector contains the herpes simplex virus VP16 activation domain upstream of a multiple cloning region. The two genes encoding the two potentially interactive proteins of interest are cloned into pBIND and […]
In vitro translation of proteins through cell-free expression systems using rabbit reticulocytes, E. coli S30, or wheat germ extracts can be invaluable in studying protein function. If you only need a small amount (100s of nanograms), it’s also faster and easier than synthesizing vast quantities in bacterial or mammalian cells (~ 90 minutes for cell-free […]
Back in graduate school, I purified a lot of RNA, and after a while, I became fairly successful at it. My yields were good, and the RNA was intact. However, many of my early attempts at RNA isolation yielded degraded RNA that did not work well in many downstream applications. In my case, successfully isolating […]
We recently posted a blog about Proteinase K, a serine protease that exhibits broad cleavage activity produced by the fungus Tritirachium album Limber. It cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples. In that previous blog we focused […]
There are a lot of choices when it comes to reverse transcriptases. Choosing the correct one for your cDNA synthesis and RT-PCR project is important. Here are a few questions that will lead you to right RT for your application:
