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NEW YORK (Reuters Health) - Doctors often assume they're explaining things in a way patients understand. When patients are confused, doctors don't always realize it. A new study shows patients might benefit from having their "health literacy" tested.
Researchers at a large Arizona healthcare center looked at how a short health literacy test would affect the way patients felt about their treatment. They found it did no harm and may have helped to improve patient satisfaction.
"The main benefit of doing health literacy assessments is simply to make clinicians aware that there are patients with limited health literacy in their practice," said lead author Ian Komenaka at the Maricopa Medicine Center in Phoenix, Arizona.
"Most clinicians assume patients understand everything but this is frequently not the case. Patients may not realize they don't quite understand, or even when they know they don't understand, they are afraid or ashamed to tell the clinician," he told Reuters Health in an email.
Health literacy - the ability to understand and use health information - depends on individual skills and also on the complexity of the health information being presented.
People with lower health literacy, for example, might not know how to take their medications or why they have to follow specific doctor's instructions.
In 2003, a U.S. government study found that 89 million Americans had limited health literacy skills and they came from all segments of society.
"Patients with low or limited health literacy have poor outcomes from a number of disease processes which results in increased costs to society," Komenaka said.
But the tests have to be short and simple, so that patients don't feel badly about being tested or not performing well.
Health literacy tests aren't often done because most of the available ones take on average 8 to 22 minutes, Komenaka said. In addition, the tests can make patients feel ashamed when they don't know the answers.
When patients feel bad about these assessments, they might feel bad about the whole patient experience, or at least that's the concern of most clinics. Previous studies indicate patients will accept health literacy assessments, but those studies were all small and did not include minorities, Komenaka and his colleagues write in the journal Surgery.
The team wanted to know if shorter health literacy tests could be done in a busy clinic and how they would impact patient satisfaction. They used an assessment tool called the Newest Vital Signs (NVS), which only takes an average of two minutes.
For two years, all patients seen at a Phoenix breast surgery clinic were given the test during the course of their routine medical visits.
They were shown a nutrition label, just like the ones on most packaged foods. Then an interviewer asked them five questions about that label in either Spanish or English, depending on the patient's preference. One point was given for each correct answer. Total scores of 4 - 6 indicated adequate health literacy and scores below 4 meant possible limited health literacy.
A total of 2,025 women took part in the study and 391 participants had scores of 4 or higher. More than half - 1,229 women - scored between 0 and 1 and 405 women had a score of 2 or 3.
In separate surveys, patients were also asked to rate their overall satisfaction with their visits to the clinic on a scale of 1 to 5, both during the year before the NVS assessment was used and during the two-year study period. The average satisfaction rating the year before the study was 3.7. During the first year the assessments were used, the ranking rose slightly to 3.8, and went up to 4.1 the second year.
These results indicate the assessment procedure did not appear to have a negative impact on patient satisfaction, according to Komenaka's team.
The study did not determine why patient satisfaction actually rose, or whether clinicians changed their behavior with patients as a result of the testing.
But in 2011, the study's second year, the clinic made a formal effort to better communicate with patients with literacy scores between 0 and 2. These included using diagrams and informational videos, instead of text, to explain how treatments and procedures like biopsies would be done. Special efforts were also made to repeat key "take home" messages and information while talking with patients.
Because the study included only women, it's not clear if the results would be similar for men, Komenaka's team cautions.
According to Cindy Brach of the Agency for Healthcare Research and Quality, addressing health literacy can be valuable, but the tests shouldn't make doctors overconfident.
"We know that people with limited health literacy are less likely to use preventive services and manage their conditions and more likely to have unnecessary hospital admissions or visits to the emergency department," Brach told Reuters Health in an email.
"Everyone benefits from clear communication and we can't really tell - even with assessments - who is not understanding at any given time. My concern with assessment is that then health care providers think those who test as adequate health literacy are always understanding, when this is unlikely to be the case," Brach said.
SOURCE: bit.ly/1cfTsGn Surgery, online October 14, 2013.


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Green & White Tea Body Scrub
Organic and naturally scented body scrubs are formulated with fine grain Dead Sea Salt, Himalayan Pink salt, and/or Bora Bora White Sand or Tahitian Black Sand. Salts are mineral-rich with Magnesium, Potassium, Bromide and Calcium. They alleviate dry skin disorders, stimulate circulation, hydrate the skin, increase moisture retention, promote cellular regeneration, detoxifies the skin, helps heal dry, scaling, irritated skin, reduces inflamation and relaxes muscles and joints, and relieves pain and soreness. Brown Sugar gently cleanses the skin for even tone, increases circulation, eases dry and itchy skin, and tightens pores while adding moisture to skin.
Organic & natural Mediterranean Sea Salts and/or Sand, Sunflower Oil, Organic Brown Sugar, Vegetable Glycerine, Castile Soap, Blueberry or Raspberry Seeds, Adzuki Bean, Ginseng Powder, Ground Luffa & Pumice, Keratin Clay, Organic natural dye, Organic & premium fragrance oils.
The combination of earthy green & slightly sweet white tea is intoxicating. Size 8 oz.
*Use only 1-2 times per week. Do not use on open wounds or face. If adverse reaction, discontinue.
- Item #: BS13 Green & White Tea Scrub

subset: commoncrawl


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Career Center
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February 5, 2013
great lengths
Outfit Details:
striped top – ModCloth
skirt – Topshop
hat – Goorin Bros
Seychelles glitter platforms – c/o Seychelles
red duffle – c/o Coach
ring – gift from my mom
lipstick – MAC Russian Red (with a darker lip liner, blended)
Since I cut my hair, I've had a lot of people ask me why I did it. Well, there are two reasons: 1) I wanted a change, and 2) it's just hair. It will grow back! I'm sure I'll miss my long hair from time to time but right now, I'm kind of loving the ease of short hair. Fickle me, I never can stick with one thing for too long.


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I've spent the past week eating that hearty miso soup I talked about earlier. It's my new diet of sorts. But with any tasty broth you're good for an hour until your stomach begs for more. "Meat!" it screamed tonight. That's what led me and Mikey to 8 oz. Burger, the new joint on Melrose Avenue by chef Govind Armstrong. Three days have passed since its opening and Armstrong just so happened to be enjoying a drink at the table next to us. With his chair facing the door, he undoubtedly had an eye on people walking in.
Mikey was the one to notice him but only later put a name to the face. If only I had known, I would've been more discreet about doing away with the top of my grass-fed burger -- eating the rest with a fork and knife.
Don't get me wrong, Armstrong has a good thing going here. As he envisioned, it's a great meal without the pretense. The patty was juicy, the onion rings, though scarce, were nicely battered, and the Kennebec fries were amazing. (Go ahead and splurge on the homemade ketchup for $1. It's worth it.). It's just that darn bun, over-sized and over-toasted, was trying to steal everyone's thunder. Mikey didn't mind, though. He rather liked it.
The burger comes medium-rare, i.e., pretty darn red, so be sure to request that it be cooked longer if you're like me with raw-meat issues. For the more faint-hearted, they also have a turkey and veggie burger options. All burgers are between $9-$11 but are fairly basic. Extra toppings — red onion marmalade, garlic roasted tomatoes, avocado, wild baby arugula and more — are $2 each.
I'd second the motion with CH-ers in speculating that as unpolished as things may seem here in these first few weeks, they'll eventually work things out. I'm rooting for them.
8 Oz. Burger Bar
7661 Melrose Ave.
Los Angeles, CA
(323) 852-0008
Their menu, courtesy of EaterLa.
Monday, July 21, 2008
8 oz. Burger (Melrose)
3 comments:
- Matthew K. said...
nice review, i'll stop by the place one day. i always love a good burger. nice pics too.
i'm wondering how the grass-fed beef tastes compared to standard corn-fed.
- Gabe T said...
Grass-fed beef is generally leaner but a lot more tasty (and healthy) than standard corn-fed.
- TonyC said...
looks like the bun has changed since my opening day post: http://chowhound.chow.com/topics/539793
but still, the grass fed version was just too thin of a patty to have an beefy impact. 25 Degrees, Bar Marmont, heck, even the Bowery's burger is better. And the noise!!!
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Excerpt from: Russell, Henry B. . "Unchanged from Colony Days." Good Housekeeping July 1907: 513-518. Google Books. Web. 6 June 2010.
Housekeeping according to the usual conception of it, is that estimable art which, by contributing in various effective ways to neatness, order, coziness, and unobtrusive elegance, produces the choicest type of a home. The word housekeeping, however may have another significance seldom used or thought of, for the good reason that the precise conditions to which it is applicable are extremely rare, at least in this country, where the rapid development of a rather migratory people has been attended by frequent moving days and constant casting aside of the old for the new. Housekeeping, in this other and rarer significance means, not simply keeping a house neat, orderly, cozy and elegant, but keeping it just as it has been kept for over a hundred years, and keeping in it, in the same places, and using daily, those things which have been used by the family for a century, some of them for over two centuries.
It applies only to that house in which every day the grandmother of today sits down to the same dining table at which her grandparents sat when they came into the house not long after their honeymoon trip of over a century ago, and, indeed, sits in the very same chairs that they sat in and the same that their ancestors sat in a century before that, or in those early colonial times when those who battled with the hard conditions of life either made great characters or died young. This kind of housekeeping therefore is that which not only keeps a house as it ought to be kept, but keeps it as it has always been kept.


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Archives and Catalogues
The following Archives and Catalogues can be helpful for proposal preparation and for preparing an observing run.
- ESO Science Archive Query Form.
For a comprehensive overview of the on-line ESO/ST-ECF archives and catalogues please refer to the Science Archive Facility.
- ESO Online Digitized Sky Survey (DSS)
- Interactive 2MASS Image Service for the NASA/IPAC Infrared Sciene Archive
Catalogues and Databases
- Standard Star Catalogue Search Tool
- HST Guide Star Catalogue (GSC)
- ESO/ST-ECF USNO-A2.0 Catalogue Server
Standard Stars Data
Below follows a non-exhaustive list of on-line standard star catalogues. Note that the information might note be fully up-to-date and that some further improvements may be needed.
- Landolt's Standard Fields, with finding charts scanned
- The General Catalogue of Photometric Data (GCPD)
- Optical and UV Spectrophotometric Standards
- Spectrophotometric Calibration OBs for EMMI (gZipped PostScript, 1.6 Mb)
- Photometric Standard Stars: NICMOS and ARNICA
- Spectrophotometric Standards
- Mid-IR spectrophotometric standard stars
- List of InfraRed Telluric Standards
- Sky map of InfraRed Spectrophotometric standard
- UVESPOP: A Library of High-Resolution Spectra of Stars across the Hertzsprung-Russell Diagram
- Siebenmorgen MidIr Standards, and Siebenmorgen/Sterzik list
Adaptive Optics Guide Stars
Guide Star Finder: A Web tool aimed at searching for suitable adaptive optics guide stars, either for single objects as for a list of observing targets.
The tool is based on the USNO-SA2.0 catalogue. Work is in progress to use USNO-B catalogue as an option. The tool also offers the possibility to choose the selection criteria from a short list of different strategies.
Optical/Infrared Interferometry
Optical/Infrared Interferometry
Please check the Registry of stars used for optical/infrared interferometry which have proven poor calibrators.


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Reviewed by Hope W. (age 12)
I really enjoyed this book. It was very funny and genuine. Samantha was funny because of her attitude and her "protests" like wearing all black. This book was very well-written. It goes into great depth about how we look at things in life and has great characterization. Samantha changed and grew in the book. By the end, she looked at art and love very differently. I felt this book was easy to relate to because it isn't perfect like a Cinderella story. It describes how life really is and is a very positive story.
I recommend this book because it has a positive story line. Lots of teenage girls would like this book because it is humorous but realistic. The plot would hold the interest of readers because it makes you laugh and think. I would also recommend this book to anyone who liked reading The Princess Diaries because it is by the same author.


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This website is sponsored by AMT Electrosurgery
Surgical plume can carry dangerous bacteria and viruses, including HIV.
As such, they can produce upper respiratory irritation, and have
in-vitro mutagenic potential.
More than 500,000 healthcare workers are exposed to laser or electrosurgical smoke each year in North America, including surgeons, nurses, anesthesiologists, and surgical technologists. When the Smoke Evacuation System is turned on, everyone can Breathe Easy. Want to Clear the Air of Surgical Plume?
Take charge of your work environment.


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KellyROlsen wrote:Here's a video I made showing a mother and father giving their son his first driving lesson in york their LEAF.
A whole generation of drivers are coming up who will have the option of never having to deal with hassles and environmental issues associated with gas vehicles.
http://www.youtube.com/watch?v=2_HtcTPY ... LfdpVj-Nww" onclick="window.open(this.href);return false;
KellyROlsen wrote:...and felt really slow on accretion. ...


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Home And Garden
Home And Garden
Home And Garden is free HD wallpaper. This wallpaper was upload at March 1, 2019 upload by admin in Home Garden.
Home And Garden is high definition wallpaper and size this wallpaper is 1280x853. You can make Home And Garden For your Desktop Background, Tablet, Android or iPhone and another Smartphone device for free. To download and obtain the Home And Garden images by click the download button below to get multiple high-resversions.Tags :
Pictures gallery of Home And Garden
DISCLAIMER: This image is provided only for personal use. If you found any images copyrighted to yours, please contact us and we will remove it. We don't intend to display any copyright protected images.


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For a list of currently available 396 projects, please follow this link. You may also browse the menu at left (or at top, for mobile phone browsers) for the current list of "396" projects for the Winter 2018-2019 semester, including both taken and open/available projects. Other projects may be added; check back if interested.
If you are looking for the BASC 396 undergraduate research project courses, please start here instead. (BASC 396 is an independent research project course which integrates both an Arts discipline and a Science discipline, open to students in any B.A. & Sc. program.)
Students interested in a potential project (that has already been submitted by a professors and already posted here) should communicate with the instructor proposing the project. Instructions on how to apply are indicated on each project description.
To post, create, or register for a new project (i.e., one that is not yet posted here), please use this online form. Students may ask their proposed supervisors to fill out the form for a project they have already agreed upon, or professors may fill out "empty" projects where they are looking for students. In other words: If you don't see a vacant project here that you like, you can make your own. Just talk to a professor, fill out the form, and follow the instructions.
See also:
- Introduction to '396' courses - general information including prerequisites and eligibility
- Information for professors
- How '396' courses work


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The process of introducing nucleic acids into eukaryotic cells by nonviral methods is defined as transfection. Using various chemical, lipid or physical methods, this gene transfer technology is a powerful tool to study gene function and protein expression in the context of a cell. Development of reporter gene systems and selection methods for stable maintenance and expression of transferred DNA have greatly expanded the applications for transfection. Assay-based reporter technology, together with the availability of transfection reagents, provides the foundation to study mammalian promoter and enhancer sequences, trans-acting proteins such as transcription factors, mRNA processing, protein:protein interactions, translation and recombination events (Groskreutz and Schenborn, 1997).
Transfection is a method that neutralizes or obviates the issue of introducing negatively charged molecules (e.g., phosphate backbones of DNA and RNA) into cells with a negatively charged membrane. Chemicals like calcium phosphate and DEAE-dextran or cationic lipid-based reagents coat the DNA, neutralizing or even creating an overall positive charge to the molecule (Figure 12.1). This makes it easier for the DNA:transfection reagent complex to cross the membrane, especially for lipids that have a "fusogenic" component, which enhances fusion with the lipid bilayer. Physical methods like microinjection or electroporation simply punch through the membrane and introduce DNA directly into the cytoplasm. Each of these transfection technologies is discussed in the following sections.
This chapter covers general information on transfection techniques and considerations for transfection efficiency and optimization. In addition, we discuss various transfection agents available from Promega as well as general protocols for transfection and specific examples using our transfection reagents. Finally, we review stable transfection and outline a protocol using drug selection.
One of the first chemical reagents used to transfer nucleic acids into cultured mammalian cells was DEAE-dextran (Vaheri and Pagano, 1965; McCutchan and Pagano, 1968). DEAE-dextran is a cationic polymer that tightly associates with negatively charged nucleic acids. An excess of positive charge, contributed by the polymer in the DNA:polymer complex, allows the complex to come into closer association with the negatively charged cell membrane. Uptake of the complex is presumably by endocytosis. This method successfully delivers nucleic acids into cells for transient expression; that is, for short-term expression studies of a few days in duration. However, this technique is not generally useful for stable or long-term transfection studies that rely upon integration of transferred DNA into the chromosome (Gluzman, 1981). Other synthetic cationic polymers have been used to transfer DNA into cells, including polybrene (Kawai and Nishizawa, 1984), polyethyleneimine (Boussif et al. 1995) and dendrimers (Haensler and Szoka, 1993; Kukowska-Latallo et al. 1996).
Calcium phosphate co-precipitation became a popular transfection technique following the systematic examination of this method in the early 1970s (Graham and van der Eb, 1973). The authors examined the performance of various cations and effects of cation concentration, phosphate concentration and pH on transfection. Calcium phosphate co-precipitation is widely used because the components are easily available and inexpensive, the protocol is easy-to-use, and it is effective with many different types of cultured cells. The protocol involves mixing DNA with calcium chloride, adding this in a controlled manner to a buffered saline/phosphate solution and allowing the mixture to incubate at room temperature. The controlled addition generates a precipitate that is dispersed onto the cultured cells. The precipitate is taken up by cells via endocytosis or phagocytosis. Calcium phosphate transfection is routinely used for both transient and stable transfection of a variety of cell types. In addition, calcium phosphate appears to provide protection against intracellular and serum nucleases (Loyter et al. 1982).
However, calcium phosphate co-precipitation is prone to variability and is not suited for in vivo gene transfer to whole animals. In addition, small pH changes (± 0.1) can compromise the efficacy of calcium phosphate transfection (Felgner, 1990). Promega offers the calcium phosphate reagent as part of the ProFection® Mammalian Transfection System—Calcium Phosphate (Cat.# E1200).
The term "liposome" refers to lipid bilayers that form colloidal particles in an aqueous medium (Sessa and Weissmann, 1968). By 1980, artificial liposomes were being used to deliver DNA into cells (Fraley et al. 1980). The next advance in liposomal vehicles was development of synthetic cationic lipids by Felgner and colleagues (Felgner et al. 1987). The cationic head group of the lipid compound associates with negatively charged phosphates on the nucleic acid. Liposome-mediated delivery offers advantages such as relatively high efficiency of gene transfer, ability to transfect certain cell types that are resistant to calcium phosphate or DEAE-dextran, in vitro and in vivo applications, successful delivery of DNA of all sizes from oligonucleotides to yeast artificial chromosomes (Felgner et al. 1987; Capaccioli et al. 1993; Felgner et al. 1993; Haensler and Szoka, 1993; Lee and Jaenisch, 1996; Lamb and Gearhart, 1995), delivery of RNA (Malone et al. 1989; Wilson et al. 1979), and delivery of protein (Debs et al. 1990). Cells transfected by liposome techniques can be used for transient expression studies and long-term experiments that rely on integration of DNA into the chromosome or episomal maintenance. Unlike DEAE-dextran or calcium phosphate chemical methods, liposome-mediated nucleic acid delivery can be used for in vivo transfer of DNA and RNA to animals and humans (Felgner et al. 1995).
A lipid with overall net positive charge at physiological pH is the most common synthetic lipid component of liposomes developed for gene delivery (Figure 12.2). Often the cationic lipid is mixed with a neutral lipid such as L-dioleoyl phosphatidylethanolamine (DOPE; Figure 12.3), which can enhance the gene transfer ability of certain synthetic cationic lipids (Felgner et al. 1994; Wheeler et al. 1996). The cationic portion of the lipid molecule associates with negatively charged nucleic acids, resulting in compaction of the nucleic acid in a liposome/nucleic acid complex (Kabanov and Kabanov, 1995; Labat-Moleur et al. 1996), presumably from electrostatic interactions between the negatively charged nucleic acid and positively charged head group of the synthetic lipid. For cultured cells, an overall net positive charge of the liposome/nucleic acid complex generally results in higher transfer efficiencies, presumably because this allows closer association of the complex with the negatively charged cell membrane. Entry of the liposome complex into the cell may occur by endocytosis or fusion with the plasma membrane via the lipid moieties of the liposome (Gao and Huang, 1995). Following cellular internalization, the complexes appear in the endosomes and later in the nucleus. It is unclear how the nucleic acids are released from the endosomes and lysosomes and traverse the nuclear membrane. DOPE is considered a "fusogenic" lipid (Farhood et al. 1995), and its role may be to release these complexes from endosomes as well as to facilitate fusion of the outer cell membrane with liposome/nucleic acid complexes. While DNA will need to enter the nucleus, the cytoplasm is the site of action for RNA, protein or antisense oligonucleotides delivered via liposomes.
Promega offers the FuGENE® HD Transfection Reagent (Cat.# E2311), a novel nonliposomal transfection reagent with wide application in different cell types and low toxicity, FuGENE® 6 Transfection Reagent (Cat.# E2691, a nonliposomal formulation that transfects a wide variety of cell lines with high efficiency and low toxicity, and the TransFast™ Transfection Reagent (Cat.# E2431), which uses a polycationic head group attached to a lipid backbone structure to deliver nucleic acids into eukaryotic cells. The best transfection reagent and conditions for a particular cell type must be empirically and systematically determined because inherent properties of the cell influence the success of any specific transfection method.
Physical methods for gene transfer were developed and used beginning in the early 1980s. Direct microinjection into cultured cells or nuclei is an effective although laborious technique to deliver nucleic acids into cells by means of a fine needle (Cappechi, 1980). This method has been used to transfer DNA into embryonic stem cells that are used to produce transgenic organisms (Bockamp et al. 2002) and to introduce antisense RNA into C. elegans (Wu et al. 1998). However, the apparatus is costly and the technique extremely labor-intensive, thus it is not an appropriate method for studies that require a large number of transfected cells.
Electroporation was first reported for gene transfer studies in mouse cells (Wong and Neumann, 1982). This technique is often used for cell types such as plant protoplasts, which are difficult to transfect by other methods. The mechanism is based on the use of an electrical pulse to perturb the cell membrane and form transient pores that allow passage of nucleic acids into the cell (Shigekawa and Dower, 1988). The technique requires fine-tuning and optimization of pulse duration and strength for each type of cell used. In addition, electroporation often requires more cells than chemical methods because of substantial cell death, and extensive optimization often is required to balance transfection efficiency and cell viability. More modern instrumentation allows nucleic acid delivery to the nucleus and successful transfer of DNA and RNA to primary and stem cells.
Another physical method of gene delivery is biolistic particle delivery, also known as particle bombardment. This method relies upon high-velocity delivery of nucleic acids on microprojectiles to recipient cells by membrane penetration (Ye et al. 1990). This method is successfully employed to deliver nucleic acid to cultured cells as well as to cells in vivo (Klein et al. 1987; Burkholder et al. 1993; Ogura et al. 2005). Biolistic particle delivery is relatively costly for many research applications, but the technology also can be used for genetic vaccination and agricultural applications.
While transfection has been used successfully for gene transfer, the use of viruses as vectors has been explored as an alternative method to deliver foreign genes into cells and as a possible in vivo option. Adenoviral vectors are useful for gene transfer due to a number of key features: 1) they rapidly infect a broad range of human cells and can achieve high levels of gene transfer compared to other available vectors; 2) adenoviral vectors can accommodate relatively large segments of DNA (up to 7.5kb) and transduce these transgenes in nonproliferating cells; and 3) adenoviral vectors are relatively easy to manipulate using recombinant DNA techniques (Vorburger and Hunt, 2002). Other vectors of interest include adeno-associated virus, herpes simplex virus, retroviruses and lentiviruses, a subset of the retrovirus family. Lentiviruses (e.g., HIV-1) are of particular interest because they are well studied, can infect quiescent cells, and can integrate into the host cell genome to allow stable, long-term transgene expression (Anson, 2004).
As with all gene transfer methods, there are drawbacks. For adenoviral vectors, packaging capacity is low, and production is labor-intensive (Vorburger and Hunt, 2002). With retroviral vectors, there is the potential for activation of latent disease and, if there are replication-competent viruses present, activation of endogenous retroviruses and limited transgene expression (Vorburger and Hunt, 2002; Anson, 2004).
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With so many different methods of gene transfer, how do you choose the right transfection reagent or technique for your needs? Any time a new parameter, like a new cell line, is introduced, the optimal conditions for transfection will need to be determined. This may involve choosing a new transfection reagent. For example, one reagent may work well with HEK-293 cells, but a second reagent is a better choice when using HepG2 cells. Promega offers the FuGENE® HD Protocol Database to help identify a protocol for your cell line when using the FuGENE® HD Transfection Reagent. A drop-down menu allows you to search the database by cell line, plate type and number of cells to be transfected. The Transfection Assistant also offers a drop-down menu to select cell lines and either FuGENE® HD or FuGENE® 6 Transfection Reagent for transfection conditions. The conditions should be considered only guidelines since you may need to optimize the transfection conditions for your specific application. See Optimization of Transfection Efficiency (Section IV) and General Transfection Protocol (Section VI) for details.
Another parameter to consider is the time frame of the experiment you wish to conduct. Is it short- or long-term? For instance, determining which promoter deletion constructs can still function as a promoter can be accomplished with a transient transfection experiment, while establishing stable expression of an exogeneously introduced gene construct will require a longer term experiment.
Cells are typically harvested 24–72 hours post-transfection for studies designed to analyze transient expression of transfected genes. The optimal time interval depends on the cell type, research goals and specific expression characteristics of the transferred gene. Analysis of gene products may require isolation of RNA or protein for enzymatic activity assays or immunoassays. The method used for cell harvest will depend on the end product assayed. For example, expression of the firefly luciferase gene in the pGL4.10[luc2] Vector (Cat.# E6651) is generally assayed 24–48 hours post-transfection, whereas the pGL4.12[luc2CP] Vector (Cat.# E6671) with its protein degradation sequences can be assayed in a shorter time frame (e.g., 3–12 hours), depending on the research goals and the time it takes for the reporter gene to reach steady state. For more information on luminescent reporter genes like firefly luciferase, see the Protocols and Applications Guide chapter on bioluminescent reporters.
When performing a transient transfection, you can choose between a standard or reverse transfection protocol. In a standard transfection protocol, the cells are plated on day 1, transfected on day 2 and assayed on day 3 or 4. In a reverse transfection protocol, cells are added directly to a plate containing the transfection reagent/DNA mix and assayed on day 2 or 3. Because the cells are added directly to the DNA, this process reduces the experimental time by one day and allows for high-throughput transfection of DNA in a plate- or microarray-format. For more information on reverse transfection including a protocol, read the PubHub article on the subject.
The goal of stable, long-term transfection is to isolate and propagate individual clones containing transfected DNA that has integrated into the cellular genome. Distinguishing nontransfected cells from those that have taken up exogenous DNA involves selective screening. This screening can be accomplished by drug selection when an appropriate drug-resistance marker is included in the transfected DNA. Alternatively, morphological transformation can be used as a selectable trait in certain cases. For example, bovine papilloma virus vectors produce a morphological change in transfected mouse CI127 cells (Sarver et al. 1981).
Before using a particular drug for selection purposes, you will need to determine the amount of drug necessary to kill untransfected cells. This may vary greatly among cell types. Consult Ausubel et al. 1995 for additional information about designing experiments to test various drug concentrations and determine the amount needed to select resistant clones (i.e., generate a kill curve).
When drug selection is used, cells are maintained in nonselective medium for 1–2 days post-transfection, then replated in selective medium containing the drug. The use of selective medium is continued for 2–3 weeks, with frequent changes of medium to eliminate dead cells and debris, until distinct colonies can be visualized. Individual colonies can be isolated by cloning cylinders, selected and transferred to multiwell plates for further propagation in the presence of selective medium. Individual cells that survive the drug treatment expand into clonal groups that can be individually propagated and characterized. For a protocol to select transfected cells by antibiotics, see Aubree2 Women's Flat Taupe Easy Dark Taupe Spirit Dark Fabric 5Eq0qxt7w (Section VII).
Several different drug-selection markers are commonly used for long-term transfection studies. For example, cells transfected with recombinant vectors containing the bacterial gene for neomycin phosphotransferase [e.g., pCI-neo Mammalian Expression Vector (Cat.# E1841)] can be selected for stable transformation in the presence of the neomycin analog G-418 (Cat.# V8091; Southern and Berg, 1982). Similarly, expression of the hygromycin B phosphotransferase gene from the transfected vector [e.g., pGL4.14 [luc2/Hygro] Vector (Cat.# E6691)] will confer resistance to the drug hygromycin B (Blochlinger and Diggelmann, 1984).
An alternative strategy is to use a vector carrying an essential gene that is defective in a given cell line. For example, CHO cells deficient in dihydrofolate reductase (DHFR) gene expression do not survive without added nucleosides. However, these cells, when stably transfected with DNA expressing the DHFR gene, will synthesize the required nucleosides and survive (Stark and Wahl, 1984). An additional advantage of using DHFR as a marker is that gene amplification of DHFR and associated transfected DNA occurs when cells are exposed to increasing doses of methotrexate, resulting in multiple copies of the plasmid in the transfected cell (Schimke, 1988).
Plasmid DNA is most commonly transfected into cells, but other macromolecules can be transferred as well. For example, short interfering RNA (siRNA; Hong et al. 2004; Snyder et al. 2004; Klampfer et al. 2004), oligonucleotides (Labroille et al. 1996; Berasain et al. 2003; Lin et al. 2004), RNA (Shimoike et al. 1999; Ray and Das, 2004) and even proteins (Debs et al. 1990; Lin et al. 1993) have been successfully introduced into cells via transfection methods. However, conditions that work for plasmid DNA transfer will likely need to be optimized when using other macromolecules. In all cases, the agent transfected needs to be of high quality and relatively pure. Nucleic acids need to be free of proteins, other contaminating nucleic acids and chemicals (e.g., salts from oligo synthesis). Protein should be pure and in a solvent that is not detrimental to cell health. For additional information on plasmid DNA quality, see DNA Quality and Quantity (Section III.D).
After cells are transfected, how will you determine success? Plasmids containing reporter genes can be used to easily monitor transfection efficiencies and expression levels in the cells. An ideal reporter gene product is one that is unique to the cell, can be expressed from plasmid DNA and can be assayed conveniently. Generally, reporter gene assays are performed 1–3 days after transfection; the optimal time should be determined empirically. For a discussion of luminescent reporter gene options, see the Protocols and Applications Guide chapter on bioluminescence reporters. A direct test for the protein of interest, such as an enzymatic assay, may be another method to assess transfection success.
In the case of siRNA, success may be measured using a reporter gene or assaying mRNA (e.g., RT-PCR) or protein target levels (e.g., Western blotting).
If multiple assays will be performed, make sure the techniques you choose are compatible with all assay chemistries. For example, if lysates are made from transfected cells, the lysis buffer used ideally would be compatible with all subsequent assays. In addition, if cells are needed for propagation after assessment, make sure to retain some viable cells for passage after the assay.
With any transfection reagent or method, cell health, degree of confluency, number of passages, contamination, and DNA quality and quantity are important parameters that can greatly influence transfection efficiency. Note that with any transfection reagent or method used, some cell death will occur.
Cells should be grown in medium appropriate for the cell line and supplemented with serum or growth factors as needed for viability. Contaminated cells and media (e.g., contaminated with yeast or mycoplasma) should never be used for transfection. If cells have been compromised in any way, discard them and reseed from a frozen, uncontaminated stock. Make sure the medium is fresh if any components are unstable. Medium lacking necessary factors can harm cell growth. Be sure the 37°C incubator is supplied with CO2 at the correct percentage (usually 5–10%) and kept at 100% relative humidity.
If there are any concerns about what type of culture medium or CO2 levels are needed for your cell line of interest, consult the American Type Culture Collection [ATCC] web site.
Silver Black Pro TD Football Vapor Metallic Low Cleats Nike As a general guideline, transfect cells at 40–80% confluency. Too few cells will cause the culture to grow poorly without cell-to-cell contact. Too many cells results in contact inhibition, making cells resistant to uptake of foreign DNA. Actively dividing cells take up introduced DNA better than quiescent cells.
Keep the number of passages low (<50). In addition, the number of passages for cells used in a variety of experiments should be consistent. Cell characteristics can change over time with immortalized cell lines, and cells may not respond to the same transfection conditions after repeated passages, resulting in poor expression.
Plasmid DNA for transfections should be free of protein, RNA, chemical and microbial contamination. Suspend ethanol-precipitated DNA in sterile water or TE buffer to a final concentration of 0.2–1mg/ml. The optimal amount of DNA to use in the transfection will vary widely, depending on the type of DNA, transfection reagent, target cell line and number of cells.
You will need to optimize specific transfection conditions to achieve the desired transfection efficiencies. Important parameters to consider are the charge ratio of cationic lipid transfection reagent to DNA, amount of transfected nucleic acid, length of time cells are exposed to the transfection reagent and presence or absence of serum. Reporter genes are useful to determine optimal conditions. The transfection efficiency achieved using any transfection reagents varies depending on the cell type being transfected and transfection conditions used.
The amount of positive charge contributed by the cationic lipid component of the transfection reagent should equal or exceed the amount of negative charge contributed by the phosphates on the DNA backbone, resulting in a net neutral or positive charge on the multilamellar vesicles associating with the DNA. Charge ratios of 1:1 to 2:1 TransFast™ Reagent:DNA have worked well with various cultured cells, but ratios outside of this range may be optimal for other cell types or applications. See the TransFast™ Transfection Reagent Technical Bulletin #TB260 for more details.
The optimal amount of DNA or RNA will vary depending on the type of nucleic acid, number of cells, culture dish size and target cell line used. For example, HEK-293 cells are optimally transfected with 0.25µg of pGL3-Control Vector (Fringe Red Strap Flat TAOFFEN Ankle Women Summer Lace Sandals Gladiator Slingback 4xqttfTOw) using TransFast™ Reagent at a 2:1 ratio in a 24-well plate. In contrast, the same cells are optimally transfected with 0.55µg of DNA using the FuGENE® HD Transfection Reagent at a 3:1 ratio in the same well size. For other cell lines, we suggest testing the DNA amounts given in Table 12.1.
Increasing the quantity of transfected DNA significantly may not yield better results. In fact, if initial transfection results are satisfactory, a reduced DNA quantity can be tested (while keeping the optimal reagent:DNA ratio constant). Often a range of DNA concentration is suitable for transfection. However, if the DNA concentration is below or above this range, transfection efficiencies will decrease. If there is too little DNA, the experimental response may not be present. If there is too much DNA, the excess can be toxic to cells. Calibrate the system using a test plasmid with reporter gene function.
Traditionally, transfection reagents must be in contact with cells for some period of time, then additional medium is added or the medium is replaced to help minimize toxic effects of the reagent. The optimal transfection time depends on the cell line, transfection reagent and nucleic acid used. For the FuGENE® HD Transfection Reagent, which is one of the more gentle methods of DNA transfection into cells, there is no need to add additional medium or replace the medium after transfection.
For initial tests with liposomal reagents that require adding or replacing the medium, use a one-hour transfection interval, and test transfection times of 30 minutes to 4 hours (Figure 12.4) or even overnight, depending on the reagent used. Monitor cell morphology during the transfection interval, particularly when cells are maintained in serum-free medium because some cell lines lose viability under these conditions. The transfection time with the TransFast™ Reagent is usually significantly shorter than that required with other cationic lipid compounds and can be decreased to as little as 30 minutes with certain cell lines. In addition to saving time, this shortened transfection time may significantly reduce the risk of cell death.
Transfection protocols often require serum-free conditions for optimal performance because serum can interfere with many commercially available transfection reagents. The TransFast™ and FuGENE® HD Transfection Reagents can be used in transfection protocols in the presence of serum, allowing transfection of cell types or applications that require continuous exposure to serum (e.g., primary cells). Note that best results are obtained when variability is minimized among lots of serum.
While many people use a single reporter gene for their experimental system, a dual-reporter system has distinct advantages. A second reporter gene allows expression to be normalized for transfection efficiency and cell number. Small perturbations in growth conditions for transfected cells can dramatically affect gene expression. A second reporter helps to determine if the effects are due to the treatment of the cells or a response from the experimental reporter.
The Dual-Glo™ Luciferase Assay System (Cat.# E2920, E2940, E2980) is an efficient means of quantitating luminescent signal from two reporter genes in the same sample. In this system, firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferase activities are measured sequentially from a single sample in a homogeneous format. In the Dual-Glo™ System, both reporters yield linear assay responses (with respect to the amount of enzyme) and exhibit no endogenous activity in experimental host cells. In addition, the extended half-life of the reporter signals are ideal for use with multiwell assay formats.
The various Promega Renilla luciferase vectors can be used as control vectors when co-transfected with a firefly luciferase vector into which the promoter of interest is cloned. Alternatively, the firefly vector may be used as the control vector and the Renilla luciferase vector as the experimental construct. In a co-transfection experiment, it is important to realize that trans effects between promoters on co-transfected plasmids can potentially affect reporter gene expression (Farr and Roman, 1992). This is primarily of concern when either the control or experimental reporter vector or both contain very strong promoter/enhancer elements. The occurrence and magnitude of such effects will depend on several factors: 1) the combination and activities of genetic regulatory elements present on the co-transfected vectors; 2) the amount and relative ratio of experimental vector to control vector introduced into cells; and 3) the cell type transfected.
To help ensure independent genetic expression of experimental and control reporter genes, preliminary co-transfection experiments should be performed to optimize both the vector DNA amount and ratio of co-reporter vectors. Because the Promega Renilla luciferase vectors are designed for optimal expression, it is possible to use very small quantities of these vectors to provide low-level, constitutive co-expression of Renilla luciferase activity. This means that the ratio of firefly and Renilla luciferase vectors to test can range from 1:1 to 100:1 (or greater) to determine the optimal expression. The key to a dual-reporter system is to maximize expression of the experimental reporter while minimizing that of the control reporter. However, the expression level of the control reporter should be three standard deviations above background to be significant.
Additionally, experimental treatments may sometimes undesirably affect control reporter expression. This compromises the accuracy of experimental data interpretation; typically this occurs through sequences in the vector backbone, promoter or reporter gene itself. For this reason, Promega offers different promoter elements with either the same vector backbone, such as that of the pGL4.7 Vector series, or a choice of vector backbones, available with the synthetic Renilla luciferase vectors (phRL and phRG), to select the most reliable co-reporter vector for your system. In fact, due to extremely complicated cellular experimental conditions, testing several vectors is sometimes required before finding the best internal control for a particular experimental situation.
The strength of the promoter in your cell system is an important consideration. A more moderately expressing promoter like thymidine kinase [TK; e.g., pGL4.74[hRluc/TK] Vector (Cat.# E6921)] may be preferable to SV40 or CMV. Stronger promoters may exhibit more trans effects, cross-talk or regulatory problems. However, adjusting the ratio of experimental vector to control vector (e.g., using 100:1 or 200:1) may eliminate some of these issues.
For a discussion of other dual-reporter assays and vector offerings, see the Protocols and Applications Guide chapter on bioluminescent reporters and the complete listing of Promega vectors.
The following sections discuss the various Promega transfection reagents.
The ProFection® Mammalian Transfection System— Calcium Phosphate (Cat.# E1200) is a simple system containing two buffers: CaCl2 and HEPES-buffered saline. A precipitate containing calcium phosphate and DNA is formed by slowly mixing a HEPES-buffered phosphate solution with a solution containing calcium chloride and DNA. The DNA precipitate is distributed onto eukaryotic cells and enters cells through an endocytic-type mechanism. Calcium phosphate transfection may be used to produce long-term stable transfectants. It also works well for transient expression of transfected genes and can be used with most adherent cell lines.
Special Usage Notes:
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MC3T3-E1 (mouse preosteoblast) cells were plated at 5,000 cells/cm² in 100mm dishes the day before transfection. Cells were co-transfected with 15µg of plasmids expressing either wildtype or dominant negative bone morphogenetic protein-2 (BMP-2) and pSV-ß-Galactosidase Control Vector at a 10:1 ratio in the presence of serum for 16 hours using the ProFection® Mammalian Transfection System—Calcium Phosphate. After the incubation, the medium was changed and the expression assessed 72 hours post-transfection by counting the number of ß-galactosidase-positive cells and assessing the expression of BMP-2 by Western blot analysis.
Drosophila S2 cells were transfected with a U6 maxigene plasmid and a control firefly luciferase construct (derived from the pGL2 Basic Vector) using the ProFection® Mammalian Transfection System—Calcium Phosphate. Expression of the maxigene was confirmed by isolating total RNA and performing primer extension analysis with the aid of the Primer Extension System. Luciferase assays were performed on an aliquot of cells after lysis with Reporter Lysis Buffer.
The TransFast™ Transfection Reagent (Cat.# E2431) comprises the synthetic cationic lipid, (+)-N,N [bis (2-hydroxyethyl)-N-methyl-N- [2,3-di(tetradecanoyloxy) propyl] ammonium iodide (Figure 12.5), and the neutral lipid DOPE.
The TransFast™ Reagent is supplied as a dried lipid film that forms multilamellar vesicles upon hydration with water. The TransFast™ Transfection Reagent delivers nucleic acid into eukaryotic cells in vitro and in vivo (Bennett et al. 1997) and performs well with many cell lines including NIH/3T3, CHO, HEK-293, K562, PC12, Jurkat and insect Sf9 cells. The TransFast™ Reagent combines the advantages of cationic liposome-mediated transfection with speed and ease-of-use to transfect cells for transient and stable expression.
TB260 TransFast™ Transfection Reagent Technical Bulletin
To explore further the role of C/EBPb isoforms in regulating p53 expression during the cell cycle, the 1.7kb murine p53 promoter was cloned into the pGL3-Basic Vector. Using TransFast™ Reagent, Swiss3T3 and 6629 (C/EBPb-null) cells were transfected using 0.1–0.75µg of pGL3-1.7-kb p53 promoter construct with or without co-transfection of 0.25µg of C/EBPb-2 and 50ng of pRL-TK Vector as an internal control. Twenty-four hours post-transfection, cells were harvested and assayed for luciferase activity, normalizing reporter activity to Renilla luciferase. Site-directed mutagenesis was used to mutate or delete the –972/–953 cis-acting element carrying the C/EBPb-binding site within the p53 promoter, and 0.1–0.75µg of each mutant construct was transfected into Swiss3T3 cells with or without co-transfection of 0.25µg of C/EBPb-2 and 50ng of pRL-TK Vector. The cells were harvested 24 hours post-transfection and assayed for reporter activity, normalizing to pRL-TK Vector activity.
The authors investigated the effect of various polymorphisms in the dopamine transporter gene (SLC6A3) on susceptibility to cocaine addiction. Genotyping of various polymorphisms in cocaine abusers and control subjects revealed a potential association of the int8 VNTR with cocaine abuse. Seven alleles of the int8 VNTR were sequenced. Various allelic sequences then were cloned into a modified phRL-SV40 Renilla luciferase reporter vector and transfected into the mouse SN4741 cell line, which expresses the dopamine transporter, and the effects on reporter activity were monitored. Sequences of two alleles then were cloned into a pGL3 Promoter Vector construct and transfected into JAP cells. The cells were challenged with various amounts of cocaine or KCl and forskolin, and the effect on reporter activity was monitored. The TransFast™ Reagent was used for transfections at a 2:1 reagent:DNA ratio.
The FuGENE® HD Transfection Reagent (Cat.# E2311) is a novel, nonliposomal formulation to transfect DNA into a wide variety of cell lines with high efficiency and low toxicity. The protocol does not require removal of serum (including up to 100% serum) or culture medium and does not require washing or changing of medium after introducing the reagent:DNA complex. Additionally, the FuGENE® HD reagent supports transfection in chemically defined media and does not contain any animal derived components. For more information about transfection conditions using the FuGENE® HD Transfection Reagent, visit the FuGENE® HD Protocol Database.
TM328 FuGENE® HD Transfection Reagent Technical Manual
PubHub Reverse Transfection Using FuGENE® 6 and FuGENE® HD
PubHub Optimize Transfection of Cultured Cells
The authors transiently transfected 2 × 10⁵ HeLa cells grown on coverslips in a six-well plate using the FuGENE® HD Transfection Reagent, 2.5µg of DNA and a reagent:DNA ratio of 8:2.5.
FuGENE® HD Transfection Reagent was used to transiently transfect HeLa cells seeded on 12mm coverslips in 24-well plates. Cells were seeded at a concentration of 5 × 10⁴ cells/well and transfected after 24 hours using a 2:1 ratio of FuGENE® HD reagent to DNA (4µl of reagent, 2µg of DNA). Transiently transfected cells were used for confocal microscopy. For luciferase assays, HEK293 cells were seeded into 48-well plates at 40% confluency, and 24 hours later, FuGENE® HD reagent was used to transfect the cells with one of a variety of constructs.
The FuGENE® 6 Transfection Reagent (Cat.# E2691) is a nonliposomal reagent that transfects DNA into a wide variety of cell lines with high efficiency and low toxicity. The protocol does not require removal of serum or culture medium and does not require washing or changing of medium after introducing the reagent/DNA complex. For more information about transfection conditions using the FuGENE® 6 Transfection Reagent, visit the Transfection Assistant.
TM350 FuGENE® 6 Transfection Reagent Technical Manual
In this study, the FuGENE® 6 Transfection Reagent was used to perform transient transfection of COS-7 cells. Plasmids containing mutated or non-mutated copies of the estrogen receptor gene ESR1 were transfected together with luciferase reporter constructs containing estrogen response elements upstream of the luciferase gene. Transactivation of the estrogen response element was reduced in the mutated estrogen receptor compared with the nonmutated receptor.
In this study, MCF7 human breast adenocarcinoma cells were transfected with plasmid constructs using FuGENE® 6 Transfection Reagent. Cells were grown to 50% confluence and transfected with 2µg of DNA at a 3:2 ratio of FuGENE® 6 reagent:DNA.
Trypsinizing cells prior to subculturing or cell counting is an important technique for successful cell culture. The following technique works consistently well when passaging cells.
Materials Required:
Typically, cells are subcultured in preparation for transfection the next day. The subculture should bring the cells of interest to the desired confluency for transfection. As a general guideline, plate 5 × 10⁴ cells per well in a 24-well plate or 5.5 × 10⁵ cells for a 60mm culture dish for ~80% confluency the day of transfection. Change cell numbers proportionally for different size plates (see Table 12.2).
a This information was calculated for Corning® culture dishes.
b Relative area is expressed as a factor of the total growth area of the 24-well plate recommended for optimization studies. To determine the proper plating density, multiply 5 × 10⁴ cells by this factor.
High-quality DNA free of nucleases, RNA and chemicals is as important for successful transfection as the reagent chosen. See the Protocols and Applications Guide chapter on DNA purification for information about purifying transfection-quality DNA.
In the case of a reporter gene carried on a plasmid, a promoter appropriate to the cell line is needed for gene expression. For example, the CMV promoter works well in many mammalian cell lines but has little functionality in plants. The best reporter gene is one that is not endogenously expressed in the cells. Firefly luciferase, Renilla luciferase, click beetle luciferase, chloramphenicol aceyltransferase and ß-galactosidase fall into this category. Vectors for all five reporters are available from Promega. See the Mugara Orchid Flat Jessica Simpson Women's Vivid Ballet w0q6W4CxWB for more information on our wide array of reporter plasmids.
In previous sections, we discussed factors that influence transfection success. Here we present a method to optimize transfection of a particular cell line with a single transfection reagent. For more modern lipid-based reagents such as the FuGENE® HD Transfection Reagent, we recommend using 100ng of DNA per well of a 96-well plate at reagent:DNA ratios of 4:1, 3.5:1, 3:1, 2.5:1, 2:1 and 1.5:1. Figure 12.6 outlines a typical optimization matrix. When preparing the FuGENE® HD Transfection Reagent:DNA complex, the incubation time may require optimization; we recommend 0–15 minutes. Incubations longer than 30 minutes may result in decreased transfection efficiency. See Technical Manual #TM328.
For traditional reagents, such as the TransFast™ Reagent, we recommend testing various amounts of transfected DNA (0.25, 0.5, 0.75 and 1µg per well in a 24-well plate) at two charge ratios of lipid reagent to DNA (2:1 and 4:1; see Figure 12.7 and Technical Bulletin #TB260). This brief optimization can be performed using a transfection interval of one hour under serum-free conditions. One 24-well culture plate per reagent is required for the brief optimization with adherent cells (3 replicates per DNA amount).
A more thorough optimization can be performed to screen additional charge ratios, time points and effects of serum-containing medium at the DNA amounts found to be optimal during initial optimization studies. One hour or two hours for the transfection interval is optimal for many cell lines. In some cases, however, it may be necessary to test charge ratios and transfection intervals outside of these ranges to achieve optimal gene transfer.
Both DEAE-dextran and calcium phosphate work well with larger cell cultures (e.g., 100mm culture dish or T75 flask). General guidelines for DNA amount and time for calcium phosphate-mediated transfection are given in Table 12.3.
a If the cells are sensitive to the reagent, incubate for no more than 4 hours. Incubation time can be longer but will need to be optimized for the individual cell line.
Some transfection methods require removal of medium with reagent after incubation; others do not. Read the technical literature accompanying the selected transfection reagent to learn which method is appropriate for your system. However, if there is excessive cell death during transfection, consider decreasing time of exposure to the transfection reagent, decreasing the amounts of DNA and reagent added to cells, plating additional cells and removing the reagent after the incubation period and adding complete medium.
Many transient expression assays use lytic reporter assays like the Dual-Luciferase® Assay System (Madden Steve Men's Olive Ace Boot Ankle 7qPxqwB) or Bright-Glo™ Assay System (Cat.# E2610) 24 hours post-transfection. However, the assay time frame can vary (24–72 hours after transfection), depending on protein expression levels. Reporter-protein assays use colorimetric, radioactive or luminescent methods to measure enzyme activity present in a cell lysate. Some assays (e.g., Luciferase Assay System) require that cells are lysed in a buffer after removing the medium, then mixed with a separate assay reagent to determine luciferase activity. Others are homogeneous assays (e.g., Bright-Glo™ Assay System) that include the lysis reagent and assay reagent in the same solution and can be added directly to cells in medium. Examine the reporter assay results and determine where the greatest expression (highest reading) occurred. These are the conditions to use with your constructs of interest.
Other assays include histochemical staining of cells (determining the percentage of cells that are stained in the presence of the reporter gene substrate; Figure 12.8), fluorescence microscopy (Figure 12.9) or cell sorting if using a fluorescent reporter like the Monster Green® Fluorescent Protein phMGFP Vector (Cat.# E6421).
Assaying relative expression using the HaloTag® technology provides new options for rapid, site-specific labeling of proteins in living cells and in vitro. The ability to create labeled HaloTag® fusion proteins with a wide range of optical properties and functions allows researchers to image and localize labeled HaloTag® protein fusions in live- or fixed-cell populations and isolate and analyze HaloTag® protein fusions and protein complexes. Several ligands are available for this system with new options being added regularly. For more information on this labeling technology, see the Protocols and Applications Guide chapter on cell labeling.
Optimization for stable transfection begins with successful transient transfection. However, cells should be transfected with a plasmid containing a gene for drug resistance, such as neomycin phosphotransferase (neo). As a negative control, transfect cells using DNA that does not contain the drug-resistance marker.
Table 12.4 provides an overview of commonly used antibiotics to select and maintain stable transfectants.
The following procedure may be used to determine the percentage of stable transfectants obtained.
Note: The stained cells will not be viable after this procedure.
For further information on stable transfections, see Ausubel et al. 1995.
1X HBSS (Hanks Balanced Salt Solution)
The final pH should be 7.1.
1X Trypsin-EDTA solution
Dissolve in a calcium- and magnesium-free salt solution such as 1X PBS or 1X HBSS.
Dual-Luciferase, GloMax, HaloTag, Monster Green and ProFection are registered trademarks of Promega Corporation.
Dual-Glo, Bright-Glo, CellTiter-Fluor, ONE-Glo, Tfx and TransFast are trademarks of Promega Corporation.
Corning is a registered trademark of Corning, Inc. FuGENE is a registered trademark of Fugent, LLC. Geneticin is a registered trademark of Life Technologies, Inc. Opti-MEM is a registered trademark of Invitrogen Corporation.
Products may be covered by pending or issued patents or may have certain limitations. Please visit our web site for more information.
All prices and specifications are subject to change without prior notice.
Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.


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One of our favorite fall views, from the treehouse at Happy Valley Orchard.
If you see me around town these days, you may notice my crazed grin. You may notice an extra bounce in my step. You may notice that I appear to be missing two-thirds of my children.
This year, two of our three daughters are in preschool three days a week.
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Flared arms perched at a jaunty angle add a little pizzazz to to this chic accent chair. Personalize it in any of our hundreds of designer fabric colors and patterns to obtain the look of your dreams. Also available in our heavy wear, stain-resistant performance fabrics to add a high level of durability and a longer life for your furniture.
The 060110 Contemporary Flare Arm Accent Chair by Craftmaster is available in the Fayetteville, NC area from Bullard Furniture.
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text: The featured Key Art was created to promote Velocity's already-aired programs on digital platforms that include download to own (DLTO), video on demand (VOD), and subscription video on demand (SVOD). The goal behind each piece was to find or composite a single image that best represented the show's particular season. When creating these designs I had to keep in mind that the title treatment had to be prominent since the ads would often be viewed on small mobile devices.


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No doubt — this is a Top 5 Seattle beer event. Each of us, over here at Beer Blotter, agree that Elysian Brewing's Great Pumpkin Beer Festival is one of the can't miss events we have here in Seattle, each year.
Elysian Brewing puts on this years celebration of all things pumpkin from October 16-17, 2010. The event begins each day at 12:00 PM and goes on through the evening.
What should you expect from this event? How about 40+ pumpkin-styled beers!
Elysian will roll out 3 or 4 beer trucks and set them up in the back lot. Each truck will pour several beers, from brewers all around the country. Here is the preliminary lineup, but remember that Elysian always holds a few names back as a surprise:
From Elysian: Night Owl Pumpkin Ale, The Great Pumpkin, Dark o' the Moon Pumpkin Stout, Hansel & Gretel Ginger Pumpkin Pilsner, Kaiser Kurbis Pumpkin Hefeweizen, PK-47 Pumpkin Malt Liquor, Mr. Yuck Sour Pumpkin Ale, and a few surprises including a new brew and treats from the Elysian Cellar.
Guest Beers From: Big Time, Silver City, Snipes Mountain, Naked City, Elliott Bay, Schooner Exact, Rock Bottom, Fremont, Diamond Knot, Anacortes, Ram-Puyallup, Rogue-Issaquah, Allagash, Russian River, Iron Hill, Cambridge, Nodding Head, Jolly Pumpkin, Dogfish Head, Stewart's, Beer Valley & Laurelwood!
We hope that you caught some of these names and did a double take — we did. Jolly Pumpkin!? Russian River?! Iron Hill!! These are some of the greatest brewers around. You can expect to see some other surprises, throughout the festival.
One other thing. Did you see that gigantic pumpkin on the Elysian webpage? Dick and the guys over at Elysian brew a beer in one of these massive gourds each year. I am hopeful that this trend continues on this year.
Don't miss this event — It's one of a kind!
Filed under: Seattle, US, The Events Tagged: Elysian Brewing, elysian dark o the moon, elysian great pumpkin beer, Great Pumpkin Beer Festival, Jolly Pumpkin, night owl, Pumpkin Beer, Pumpkin beer events, pumpkin beers, pumpkin food, russian river pumpkin, seattle beer, seattle beer events


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Actress Isla Fisher has no plans to work with her funny man partner Sacha Baron Cohen because she is convinced they collaborate enough at home as busy parents.
"The Wedding Crashers" star scaled back on her career to care for her young daughters – Olive, 5, and 2-year-old Elula, but she has returned to the spotlight with two new films, "Now You See Me" and "The Great Gatsby."
With her career now back on track, the Australian beauty has ruled out co-starring in a movie with her husband as they have their hands full raising their kids.
She tells "Entertainment Tonight," "Personally, I think we collaborate on enough behind closed doors. We're working on some really important collaborations (our daughters), and I think that's more important.
"It's not like you're taking a break and looking on IMDbPro (website) to see your StarMeter falling. You're doing the most important, incredible thing. When you come back in, the perspective has changed. I truly believe you can't have it all and you shouldn't want to."
Fisher admits she still admires the comic's work, adding, "Obviously, I'm a big fan of his. My favorite movie will always be Bruno, and my favorite line will always be when he looks up at the sky and goes, 'So many stars in the sky. Makes you think of all the hot guys in the world.' So freaking random and funny."


text: Allegations of domestic violence have serious legal ramifications, while also highlighting the complicated emotional aspects of domestic abuse. Even if a couple reconciles, prosecutors may continue to pursue criminal charges against the alleged perpetrator. If you've been accused of domestic battery, stalking, harassment or other forms of domestic violence, you need the advice and counsel of a criminal defense attorney who has experience handling these types of cases. A domestic violence lawyer or law firm can also represent you if you are seeking a restraining order or order of protection, or if someone has asked the court to issue a restraining order against you.


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Louis Vuitton Red Epi Jasmin Bag Authentic Pre Owned
Authenticity code: Louis Vuitton TH1000
Authentic: Pre Owned
This authentic LV Red Epi Jasmin is gorgeous! It features LV signature epi leather with a smooth leather trim and double rolled leather handles. It has a double zip top closure and spacious interior that is lined in alcantara lining. It features stunning silver tone hardware that radiates against the red epi leather! A classic and timeless bag!
- Exterior: Gently used
- Interior: Gently used
- Description of Interior: Alcantara Lined
- Color: Red
- Hardware Description: Gold Hardware
- Material: Epi
- Width: 12.6 in.
- Length: 3.5 in.
- Height: 7.1 in.
Product Code: 2192025
Read more about Louis Vuitton Date Code here.
All photos are of actual item being sold. © 2007-2019 LuxeDH.
This Louis Vuitton® item is guaranteed to be authentic. Louis Vuitton is a registered trademark of Louis Vuitton. LuxeDH is not affiliated with or represents Louis Vuitton in any way or form.


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VCA Animal Hospitals in Hawaii is seeking experienced Veterinarians to join our team as as Associate Veterinarian in either Emergency or GP practice on the Oahu Island. This is a rare opportunity to perform top-notch medicine, surrounded by a dedicated, well-trained, friendly team of professionals in one of the most beautiful places on earth.
Join our group of progressive, growing hospitals '" including one specialty/24 hour Emergency hospital - focused on providing quality medical care with a personal touch to our community'™s pets. Our well-appointed hospitals and experienced and knowledgeable medical teams ensure our patients receive the best veterinary care in the industry! This is a dream job for an experienced General Practitioner or Emergency Doctor: established reputable hospitals with strong, loyal referral clients. We provide a comprehensive benefits package, support in building and growing your practice and the stability that is inherent in a company like VCA.
Appropriate candidates for this position should be friendly, outgoing, collaborative, enthusiastic and ready to join a fast-paced, friendly group of professionals. This is a wonderful opportunity for the right candidate. Preference given to candidates with at least 2 years of experience.
You would be hard pressed to find a more beautiful part of the country. Oahu is a gorgeous island with easy access to secluded tropical beaches, snorkeling at the well-known Hanauma Bay, world-renowned golfing, deep-sea fishing, and stunning hiking and mountain biking. On Oahu, you are guaranteed unsurpassed beauty, tropical views and cultural infusion. The historic and cultural attractions are plentiful and unmatched by the other islands.
Among the reasons to consider joining VCA are: '¢ Network of 4,500+ doctors, including more than 600 Specialists'¢ Largest provider of Private Practice Internships and Residencies in the U.S.'¢ WOOF University, offering abundant CE for Doctors and Staff'¢ Robust Clinical Studies program'¢ Opportunities to give back through strong Shelter partnerships and VCA Charities We offer competitive compensation and great benefits for eligible employees, including medical/dental, generous personal pet care discounts and more!


text:
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One Direction Live in San Diego Contest Winner Announced
Congratulations to Michelle Peck —she's the winner of our One Direction Live in San Diego contest!
Michelle and a guest will fly to gorgeous San Diego, CA to see One Direction live on August 4th. They'll also get access to the sound check before the show. That's right! They will get to see One Direction set up for the show and test out their songs before hitting the stage for real. Plus, we're giving her $500 to spend any way she wants.
Who will win next? Our VIP program has tons of great contests happening right now—including the hottest gadgets, shopping sprees, cash, trips and more. You can also play games, access exclusive content, and share your feedback.


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This is a first cam check assembly for the Wilkins 375 series reduced pressure zone assemblies. This is a complete first cam check. The check is held in place by a plastic retainer ring. Use caution when removing the retainer ring as they often break. Replacement retainer ring is included. Genuine Zurn/Wilkins repair parts. No special tools are required for servicing.


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This document describes the recommended coding style for CMake files in KDE, i.e. CMakeLists.txt files and *.cmake files.
To put in in one sentence: be as careful when writing the CMake files as when you are writing C++ code.
Indent all code correctly, i.e. the body of
Use spaces for indenting, 2, 3 or 4 spaces preferably. Use the same amount of spaces for indenting as is used in the rest of the file. Do not use tabs.
Most important: use consistent upper- or lowercasing within one file !
In general, in KDE the all-lowercase style is preferred.
So, this is recommended:
add_executable(foo foo.c)
This is also acceptable:
ADD_EXECUTABLE(bar bar.c)
Mixed casing as shown below works too, but should not be done within KDE:
Add_Executable(hello hello.c) aDd_ExEcUtAbLe(blub blub.c)
To make the code easier to read, use empty commands for endforeach(), endif(), endfunction(), endmacro() and endwhile(). Also, use empty else() commands.
For example, do this:
and not this:
if(BARVAR) some_other_command(...) endif(BARVAR)
You are free to use pkg-config in FindXXX.cmake modules, as long as the following conditions are met:
Follow the style guide from CMake when writing some FindFoo.cmake module: readme.txt
For checking the results inside the Find-module, the macro find_package_handle_standard_args() (coming with CMake) should be used, using the new extended syntax, which supports also version checking.
Micro-optimizations like
if(FOO_LIBRARY AND FOO_INCLUDE_DIR) set(FOO_FOUND TRUE) else() ... execute the whole find-logic endif()
should be removed, the find-logic should be executed always. These shortcuts can cause problems e.g. when the same file is used from multiple directories but e.g. with different required versions or components etc.
Also manually quieting the module should not be done:
if ( SQLITE_INCLUDE_DIR AND SQLITE_LIBRARIES ) set(Sqlite_FIND_QUIETLY TRUE) endif ( SQLITE_INCLUDE_DIR AND SQLITE_LIBRARIES )
If find_package_handle_standard_args() is used, this is completely unnecessary, since find_package_handle_standard_args() only prints something if the result has changed compared to the previous run. So if, as in this example, sqlite has already been found, find_package_handle_standard_args() will only print this information during the first CMake run, but not on successive runs.
So, do not add such code. It is unnecessary and makes the modules look more complicated than necessary.


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Today at Colliding Universes
Origin of life: The misnaming of the Great Oxidation Event - which maybe never happened, so forget your prof's huffing way back when
Time: Can time flow backwards in quantum physics? Maybe ...
Hubble Space Telescope: And awesome introduction
History moment: Moon landing recalled
Can the laws of physics evolve?
Colliding Universes is my blog on competing theories about our universe. You can search it via the Search Blog box at the top left, beside the "B" logo.
(Note: If you follow me at Twitter, you will get regular notice of new Colliding Universes posts, usually when I have posted five or so stories.)
Colliding Universes is my blog about competing theories of our universe.


text:
Part 1 (Book 1 - Chs 1-12). Classic Literature VideoBook with synchronized text, interactive transcript, and closed captions in multiple languages. Audio courtesy of Librivox. Read by Rebecca Dittman: http://www.mercurialspirit.co.uk/
Playlist for The War of the Worlds by H. G. Wells: http://www.youtube.com/playlist?list=PL5752CEE9CCEA7F12
The War of the Worlds free audiobook at Librivox: http://librivox.org/war-of-the-worlds-solo-by-h-g-wells/
The War of the Worlds free eBook at Project Gutenberg: http://www.gutenberg.org/ebooks/36
The War of the Worlds at Wikipedia: http://en.wikipedia.org/wiki/The_War_of_the_Worlds
View a list of all our videobooks: http://www.ccprose.com/booklist


text:
The marvellous image is part of How to Put Corner Shelves in Your House document which is grouped within Corner, Shelves, Furniture and published at October 14, 2015 2:09:05 pm by rchadmin
How to Put Corner Shelves in Your House: Built In Corner Shelves
Do you concur that corner shelves are imperative furniture in your home? You may encounter challenges when outlining the inside of the house, particularly in the corners of the room. Now and then, after deliberately put all the furniture, this range looks right. Either in light of the fact that you give the impression of space enrichment is thin or excessively exhaust.
Along these lines, you can assemble your home with an exceptionally present day request in the corner of the room. The objective is to make the figment that the corner of the room you are "grasping the room". By making a more adjusted corner shelves, as though there was no point by any means.
Corner shelves in home
Notwithstanding, in the event that you have a house fabricated in the customary way, this may not matter. Here are five approaches to enliven the corners of your home. You can utilize the rack set bended corners of the room. Corner shelves can store little statues, toys, and recollections as photos or significant articles from the spots you have gone by.
Change into a corner of the room you are perusing. This is the right thought in home. You have a window close to the corner so while understanding, you will get a characteristic shine. You can make a small library that comprises of a shelf mounted on the divider also or in a close-by rack and a seat or easy chair.
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Back to How to Put Corner Shelves in Your House


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By Emma Marsano | Special to the Almanac
Three feature films and a series of three shorts will be shown at the Windrider Film Forum at the Menlo-Atherton Performing Arts Center from Thursday through Saturday, June 19-21.
The forum shows films that "address some human issue ... in a way that encourages the audience to openly explore the issue, dialog with the filmmaker in the Q&A sessions, and (come away) more educated on the topic and interested in constructively responding to the issue," said Terri Bullock, director of the Bay Area Windrider Film Forum.
Panel discussions will follow these screenings:
● 7 p.m. Thursday, June 19: "Hank and Asha," written, directed, produced and edited by husband and wife James E. Duff and Julia Morrison. The film explores a long-distance romance between two people from different cultures and backgrounds and asks viewers to examine how people communicate in a hyper-connected world.
● 7 p.m. Friday, June 20: "Short Term 12," which won the Grand Jury Award and the Audience Award at the 2013 SXSW Film Festival. In making the film, Destin Daniel Cretton drew from his experience working at a home for at-risk teenagers.
● 2 p.m. Saturday, June 21: Three short films tackle the immigration issue from different angles: "Old Immigrant's Dance," "The Fence" and "The Dream Is Now."
● 7 p.m. Saturday, June 21: "Alive Inside: A Story of Music and Memory," a documentary that won the Documentary Audience Award at the Sundance Film Festival this year. The film tells the story of a man who volunteered at a facility for elderly people with dementia. He began playing their favorite songs for them, and watched them go from sitting quietly to singing along with family members in a matter of minutes. Producer Alexandra McDougald and filmmaker Michael Rossato-Bennett will participate in the panel discussion.
The Performing Arts Center is at Menlo-Atherton High School, 555 Middlefield Road in Atherton.
Go to tinyurl.com/Wind-619 for more information and to order tickets.


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Nostalgia Week Continues
After Wednesday's Wonder Twins adventures, we thought we'd do more comics that would be incomprehensible to half our readers. Evidence of the phenomenon seen in today's strip - and I don't mean the phenomenon where Gabe implies I might be a girl - is ubiquitous, unless you don't watch movies or read comics or play with toys or see anything at all ever. It was the new He-Man in particular that started our conversation about it, but there's another big Transformers push as well, along with other stuff I'm sure. I don't even have to look, I know it's there. For everything else we used to like, it's only a matter of time. It's a shame that their vile (but welcome!) machinations are limited only to toys and other merchandise, as it might be nice to recoup my optimism and potential while they're fucking around in the eighties. I mean, if they are taking requests, Marie from Bible Camp was pretty rad. Just see what you can do, I guess.
Our stuff from Videogame Depot is supposed to arrive along a very short timeline, and I hope to not string you along this time and actually write the things I said I was going to. It should help that I actually chose my games for this shipment, and it would be hard not to look forward to these - I mean, listen. To hear IGN tell it, this new Castlevania for the GBA I've got coming ain't bad at all. I could see at E3 that visibility was much improved in this second offering, but the expo really isn't the best place to actually play a game and I set it back down. Apparently menus are in English, but inventory items and conversations are still in Japanese - I'll let you know how much that actually gets in the way. Gabe called my other game an "earthquake sim," or whatever, but if a something isn't on the Neo-Geo he sort of zones out most of the time. He might try to deny that, but he'll stop denying it in mid-sentence and kinda stare off into space, and it might be fifteen minutes or more before he comes all the way back. Zettai Zetsumei Toshi piqued my curiosity when I heard about it on The GIA so long ago, so pointing you to their spiritual successor in order to get more information seems somehow appropriate. In the aftermath of a devastating earthquake, it sounds like we're in for "survival horror" - plenty of puzzles, and an ominous, disintegrating urban environment as your enemy. I can't wait.
A couple months ago, I made it clear that MC Frontalot was the official rapper of Penny Arcade, to single him out among all the other rap artists we are apparently affiliated with. He sent a mail inquiring as to what exactly that post entails, and I told him that being one's official rapper is much like being a Poet Laureate, only a good deal more funky. His duties would include breaking it down on occasion, busting and/or kicking it, in addition to knowing what time it is. He seemed keen to the concept, and we thought that perhaps a Penny Arcade Theme Song would be a fitting venue for his unchecked flavour. Then, I forgot all about it - until I saw a thinly veiled reference to it at his site, which has me all aflutter. I don't really have an ETA or anything. I haven't even talked to him about it. I'm just, you know, excited. I am in a state of Woo.
Hey, you've seen it, let's all look at it again: I love it when guys screw around with their consoles, make them portable, put a bunch of them in one box, give them wood panelling. This guy has, with with fell magicks, fused a GBA and that LCD screen they make for the PSOne into an unholy and unnatural marriage. Which I'm basically fine with.
(CW)TB out.
it's like the amount


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What does it take for you to feel the cadence of winter? For snowkiters in the northern Rockies I think I know just what it takes to feel that rhythm. It takes powder snow, smooth wind, endless sessions with friends and no pause button. Drive to another kite spot and repeat the next day. If I don't feel that tempo, my winter is not complete. For the past three weeks, it has been close to a perfect beat. My truck's gas bill is hitting 8 C notes a month; I know I'm in the swing of things.
Winter Solstice: The shortest day of the year delivered another high wind session. Winds were 30mph with gusts to 40 at Raynolds Pass and at The Wave in Island Park. With close to a foot of fresh cold powder it was another day to mark the cadence of winter. For me, having day after day of kiting, a little Nordic XC, some ice kiting for diversion and an occasional recovery day is what makes for contentment.
Demos: Ozone Access and Frenzy kites are everywhere I go and for most kite days I have persuaded someone to put one of these kites into the sky and go for a session. Every kiter who has taken one of this year's kites out has come back happy. Here's a few comments from the past couple weeks: "Speed: This kite is fast! Controlled and precise. Light bar pressure. The kite gave me lots of time before I needed to redirect. Handles smoothly. Loops fast! Holds its shape amazingly..." What I am not able to put to words is the smiles that these kiters have while out ripping soft snow with these superb kites. If you are in Montana, Idaho or Wyoming, be sure to hook up with me in the next few weeks.
Weather Forecast: The jet stream is delivering the goods. We have wind and more wind for the next week of weather models. It has been like that all over Montana. Yeah, a little driving to get to the next windy spot, but it's on and with the last storm, the snowpack is starting to get deep. Another dump or two and bye-bye sagebrush until May.
So for all the kiters who are making New Year's plans, drive safe, keep your cooler stocked and see ya out there!


text:
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*Business days include Monday - Friday, excluding holidays.
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Option 2. Simply use the prepaid return label included in your order, place it on the box, and drop off at your local post office. If there was an issue with your order, you will not be charged for the return shipping. If you simply changed your mind, a $7.95 restocking fee will be deducted from your refund.
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text:
Please Note: The grant information below is for historical information only. The competition is closed. Please continue to visit our site for future competition information.
Application Deadlines:
- Deadline for Transmittal of Applications: Closed February 23, 2009
- Deadline for Intergovernmental Review: Closed April 22, 2009
Current Application
Applications for grants under this competition must be submitted electronically using the Grants.gov
Through this site, you will be able to download a copy of the application package, complete it offline, and then upload and submit your application. You may not e-mail an electronic copy of a grant application to us.
For printed applications contact:
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