CRISPR/Cas9 is relatively simple to implement, as the researcher fully controls the experimental design of the tools, from the sgRNA sequence to the Cas9 protein.
We have been closely following the interesting case of the discontinuation of tobacco acid pyrophosphatase (TAP) and the efforts of the world’s RNA biologists to find a suitable alternative. Our previous post gives a bit of the background and discusses how protocols for 5′ RACE, transcriptional start site (TSS) mapping and GRO-Seq require a good decapping enzyme to remove cap structures from RNAs to allow oligocapping. Here we discuss two new commercial offerings to replace the discontinued TAP enzyme and show some user data comparing the 3 enzymes. [Read more…]
GeneCopoeia brand products include any possible DNA construct/plasmid in addition to an ever-growing offer of associated products and services. Here is a quick overview of the offer:
A few months ago I read a very nice blog post from our friends at TriLink Biotechnologies giving the chemist’s perspective on the excitement surrounding “Click Chemistry” and how it can be used to make non-natural, yet functional DNA and RNA. Some of the terms in that post such as 1,3-dipolar cycloaddition are oriented more towards chemists. Here’s a more biologist-friendly explanation of Click Chemistry: [Read more…]
July 7, 2015 marked the 20-year anniversary of the filing dates of both the U.S. patent and European patent limiting the use of PolyEthylenImine (PEI) as a transfection reagent. Coincidentally, both U.S. and European patents generally have a term of 20 years from the filing date.
Many academic researchers have been ignoring these patents and/or have been sharing protocols online and publishing articles explaining how cost-effective and simple PEI-mediated transfection can be. For example, the most commonly used PEI, catalog nr. 07923966-2 (Polysciences), is a linear form with molecular weight of 25,000 Da. The 2 gram bottle of PEI powder can be used to make a few liters of transfection reagent, so depending on which protocol is used the cost can be about 0.01% that of commercially-available transfection reagents. [Read more…]
Many labs have adopted the CRISPR genome editing technology to make knock-out and knock-in cell lines.
This technology produces first a targeted break in genomic DNA, which can then be exploited to produce cell lines with genes knocked out or where a donor vector has been used to introduce new genetic elements (point mutants, fluorescent tags, antibiotic resistance cassettes, etc.). Essentially any desired modification to the cells genome can be made. In setting up these genome editing projects there are many choices to be made including vector for the Cas9 protein and for the sgRNAs. Perhaps the most difficult choice, however, can be which cell line to use. Even the most affordable stable genome editing cell line development services can come with a significant cost, so choosing the right cell line at the beginning is crucial. Here we explain some of the choices researchers have in setting up their CRISPR genome editing projects and give our advice for cell line selection.
Molecular biologists are familiar with the QuikChange® Site-Directed Mutagenesis Kit that allows rapid intoduction of a point mutant into a plasmid/vector/mammalian expression construct. Briefly, the protocol first involves a thermocycling/PCR step with mutagenic primers, followed by a DpnI digestion step to digest the methylated parental/wild-type plasmid, and finally transformation into competent cells for nick repair.
Most experts we’ve talked to still use this technique, but don’t see the point of an expensive kit. Instead they use their own protocols with inexpensive enzymes and reagents bought separately. One such protocol involves the following: [Read more…]
Genome editing technology enabled by CRISPR and TALEN has become mainstream. Most cell biology labs are engaged in projects to create custom cell lines with knock-outs and knock-ins, and companies such as GeneCopoeia even propose complete cell line generation services. Projects can involve transfection of mammalian expression constructs, TALEN pairs, or direct transfection of RNA.
When scientists want to make a stable knockout cell line, one of the first questions they should ask is whether or not the resulting cell line will be viable. Often a murine knockout mouse has been made and/or siRNA knockdown experiments have been performed in human cells, so experienced users have a good idea if a human knockout cell line will be viable. There are certainly some cases, however, where either the researcher knows or expects that a complete knockout will not be viable but wishes to make the knockout nonetheless. What is the best way to deal with all of the risk involved with starting an relatively expensive and time-consuming project like this that could end in failure? [Read more…]
In 2012, Roux et al. published a nice paper, that received no less than four article recommendations from F1000 researchers. The paper described a method for tracking the interaction partners a protein has had within a cell (a history of its interacting partners). The method, called BioID, is based on proximity-dependent biotinylation of proteins by a promiscuous biotin ligase mutant BirA (R118G), which is fused to your protein of interest. After an overnight incubation with biotin, cells can be subjected to harsh lysis and biotinylated proteins can be isolated and identified by mass spectroscopy to determine the proteins that had come into contact with the chimeric BirA (R118G) protein. This method is a bit different from standard co-IP or pull-down experiments, because it allows one to identify proteins who interact transiently or weakly with the protein of interest. Also, due to the strong biotin-avidin binding affinity, harsh washes can greatly reduce background protein binding. [Read more…]
For decades researchers have been using the famous Alexa Fluor® dyes developed by Molecular Probes for everything from flow cytometry to oligonucleotide labeling. These sulfonated forms of common fluorophores (coumarin, rhodamine, fluorescein, and cyanine) are thought to be more stable, brighter, and less pH-sensitive than the native molecules (Ref: Panchuk-Voloshina et al. 1999). Antibody users are likely most familiar with Alexa Fluor® 488 and Alexa Fluor® 594 which when used together allow for the simultaneous staining of one protein in green and another in red and can be used in combination with the blue nuclear dye DAPI. [Read more…]