The 5 Stages of Failed Cloning Grief (and how to get back on track!)

Cloning is a fickle process that can make even the most seasoned bench scientists scream in frustration. By the time you perform a colony PCR and run the gel to check for your insert, you’ve invested several days in preparing these transformed cells. But then, the unthinkable happens. When you image your gel…the target band is missing.

This can trigger what’s known as “The 5 Stages of Failed Cloning Grief.” As you work through each stage at your own pace, just know that scientists all over the world feel your pain and can empathize with you in this difficult time. Continue reading

A Crash Course in Fighting Lab Contamination

When I first started in my undergraduate lab, one of the first things I learned was how to prepare agar plates for growing yeast. My supervisor, a grad student, looked over my shoulder as I added the yeast extract, bacto peptone, and other ingredients. I sealed the pitcher tightly with aluminum foil and autoclaved it until sterile. When I was ready to pour the plates, I carried the pitcher to the “plate-pouring” room, ripped the foil off, and started to pour an even layer of agar into each of the plastic dishes, leaving the lids off so they could cool. After I’d poured a dozen or so, my grad student supervisor burst into the room.

“What are you doing?” she demanded.

“I’m pouring plates,” I stammered back.

She took a deep breath and explained. By fully uncovering the pitcher and leaving my plates uncovered, I had left my precious media at high risk for contamination. The open containers were far too inviting for potential contaminants floating through the air. In the end, we ended up throwing away several of the plates that had been exposed the longest.

Now, I don’t share this story to demonstrate how clueless when I first started in the research lab as an undergrad. We all have those “uh-oh” moments when we realize for the first time that something that seemed so obvious was, in fact, more complicated than we’d expected. However, that day I learned how easily I could sabotage my own work by unwittingly inviting contaminants into my experiments.

Whether you work with yeast, bacteria, mammalian cells or anything else in a molecular biology lab, preventing contamination is crucial to getting desired results. Fortunately, minimizing your risk can be incredibly easy.

Let’s start with your lab bench. Everyone has their own organization system, but if yours is “out-of-control chaos,” you might want to reevaluate. Benchtop clutter makes it difficult to thoroughly clean the bench as often as needed. All those bottles of solutions, empty tip boxes, and wrinkled protocol sheets harbor dust and other unwelcome particles that you want to keep away from your cultures and reactions.

Once your benchtop is tidy (or at least somewhat tidy), make sure you keep the surface as clean as possible. Immediately clean up any spills or drips that happen while you’re working. Wiping your workspace with a 10% bleach solution will sterilize it, and following that up with 70% ethanol will dry it quickly. This wash should be performed at least once a day. Ideally you should also regularly remove everything from your workspace and perform a deeper cleaning of your benchtop, as well as any shelves and containers in your area.

Now that your bench is in good shape, it’s time to gear up . You should always follow standard safety procedures (lab coat and goggles, closed-toe shoes, hair tied back), but above all, make sure you never forget your gloves. Gloves protect you from harmful chemicals, but they also protect your experiments from anything that could be on your hands. Skin can carry reagents, bacteria, and enzymes that are good for your body but bad for your experiments. Change your gloves regularly to prevent potential carryover of reagents or samples between containers. A good rule is, “When in doubt, change your gloves.”

Finally, to guard against airborne contaminants, do your best to keep everything covered when you aren’t immdiately using it. I learned this rule the hard way when several of my yeast plates developed fuzzy patches of mold several days after I poured them. Bacteria and other undesirables floating through the air can affect stock solutions, cultures, plates, tubes, and basically anything else you rely on. Keep your lids on and cover open containers to minimize air exposure to reduce the chances of nefarious particles finding their way in.

There’s no way to guarantee you’ll never experience some form of contamination in your lab, but smart practices can help reduce your risk. Develop an anti-contamination routine that meets your needs and make sure you stick to it every day in the lab.

Working with RNA doesn’t have to be a nightmare

We’re all familiar with the Central Dogma of Molecular Biology: DNA is transcribed into RNA, which is translated into proteins. It’s drilled into our heads from the early days of biology classes, and it’s surprisingly useful when we start exploring in our own research projects. For example, if you’re interested in gene expression, you’ll most likely be working with RNA, specifically mRNA. Messenger RNA (mRNA) is transcribed from DNA and is used by ribosomes as a “template” for a specific protein. The total mRNA in a cell represents all of the genes that are actively being transcribed. So, if you want to know whether or not a gene is being transcribed, RNA purification is a great place to start.

When preparing your RNA samples for a downstream assay, there are several roadblocks and pitfalls that could give you quite a headache. Let’s tackle two of the most common.

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How to Take Care of Your Pipettes

what not to do with your pipettes

Pipettes are such a routine part of everyday life in the lab that it can be easy to take them for granted.  Their accuracy is vital, and there are many things we can adopt as best practices for success. Here are a few tips (no pun intended) gathered from around the Web by Kim Steinhauser of the Promega Metrology Department–the group charged with keeping our pipettes and other lab equipment functional and accurate. Continue reading

Will This Kit Work with My Sample Type?

Whether you are working with cells, tissues or blood—making sure you use the correct assay system is critical for success.

In Technical Services, we frequently answer questions about whether a kit will work with a particular type of sample. An easy way to find out if other researchers have already tested your sample type of interest is to search a citation database such as Pub Med for the name of the kit and your specific sample type. We also have a searchable peer-reviewed citations database on our web site for papers that specifically cite use of our products. And on many of our product pages, you can find a list of papers that cite use of those products. In Technical Services, we are happy to help you in this search and let you know if scientists here at Promega have tested a particular application or sample type. This information provides a good starting point to optimize your own experiments.

One common question is “can the Caspase-Glo® Assays be used with tissue homogenates?” While Promega has not tested the Caspase-Glo® Assays with tissue homogenates, scientists outside of Promega have used the assays with tissue homogenates with success. As with almost all of our kits, Resources are provided on the catalog page including a list of Citations. As an example, here is a link to the Citations for the Caspase-Glo® 3/7 Assay Systems. We also have an article highlighting a citation on detecting caspase activities in mouse liver. A variety of lysis buffers have been used to make tissue homogenates for this application. To avoid nonspecific protein degradation, it is useful to include a protease inhibitor cocktail in the lysis buffer. The use of protease inhibitors doesn’t usually affect our assay chemistries. Additionally, many commercially available protease inhibitor sets can be used that do not contain caspase inhibitors. It is important to consider the specificity of the kit being used and include proper controls to ensure that the luciferase reaction is performing as expected. For more information on citations and example protocols, feel free to contact us here at Technical Services and we can help get you started with your sample type.

How to Reduce Cell Culture Variability

Scenario 1: Jake needs a flask of MCF-7 cells for an assay, so he sends an email to the graduate student listserv asking for cells. Melissa replies that she has an extra flask of cells that she could share. Jake happily accepts the cells and begins his experiment.

Scenario 2: Michael passaged his cells yesterday and, according to the protocol, was supposed to plate cells today for treatment. However, his previous experiments were delayed, so he decides to plate them tomorrow instead. The cells look healthy, so it should be ok.

What is wrong with the above scenarios? These actions may seem harmless, but they could be the cause of variability, leading to irreproducible results. Continue reading

Which DNA Do I Use? How to Choose Your Control and Other DNA Samples

 

DNA double helix molecules and chromosomes.

Today’s Promega Connections blog is written by guest blogger Joliene Lindholm, Promega Technical Services Scientist.

In Promega Technical Services, we are frequently asked questions about choosing among our Human Genomic DNA products. Promega offers DNA that can serve as sources of normal human gene sequences or positive controls where genotype is not critical, and controls for use in genotyping applications like STR analysis. For mouse researchers, we also offer Mouse Genomic DNA. Continue reading

Weird samples? Contact Tech Serv to find the right DNA purification kit for you.

“Dear Tech Serv,
We would like to detect DNA collected from swabs rubbed on the inside thighs of frogs. What would be the best DNA extraction kit to use for this?”

“Hi Tech Serv,
I need to find out a suitable kit for extracting DNA from bird fecal samples. Can I use ReliaPrep™ gDNA Tissue Miniprep System for that?”

These are just some examples of unconventional sample type inquiries that the Promega Technical Services Team receives regularly from scientists around the world. Many of these inquiries land in the hands of Technical Services Scientist, Paraj Mandrekar (a.k.a. “sample type guru”). Continue reading

Optimizing Your Scientific Conference Experience

When I was in graduate school (a really long time ago), I remember going to my first big conference—American Society for Cell Biology—and being completely overwhelmed. I walked in with my Annual Conference Proceedings (back then it was all paper—no apps—and those books were thick, heavy and took up a ridiculous amount of space in your luggage). I had highlighted at least 100 posters that I was going to visit, along with one talk at every session that remotely applied to my work. And of course, I was not going to miss a single platform presentation. I was grimly determined to learn everything.

After a day-and-a-half, I was too tired to even troll the exhibition floor for freebies.

In my current job, I spend time monitoring hashtags for scientific conferences, and I occasionally notice a plaintive tweet from a conference attendee awash in a sea of posters and platform presentations—wondering where to start or where to stop.

So I asked our scientists at Promega what their tips are for getting the most out of a conference. Here are our Conference ProTips:

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Better NGS Size Selection

One of the most critical parts of a Next Generation Sequencing (NGS) workflow is library preparation and nearly all NGS library preparation methods use some type of size-selective purification. This process involves removing unwanted fragment sizes that will interfere with downstream library preparation steps, sequencing or analysis.

Different applications may involve removing undesired enzymes and buffers or removal of nucleotides, primers and adapters for NGS library or PCR sample cleanup. In dual size selection methods, large and small DNA fragments are removed to ensure optimal library sizing prior to final sequencing. In all cases, accurate size selection is key to obtaining optimal downstream performance and NGS sequencing results.

Current methods and chemistries for the purposes listed above have been in use for several years; however, they are utilized at the cost of performance and ease-of-use. Many library preparation methods involve serial purifications which can result in a loss of DNA. Current methods can result in as much as 20-30% loss with each purification step. Ultimately this may necessitate greater starting material, which may not be possible with limited, precious samples, or the incorporation of more PCR cycles which can result in sequencing bias. Sample-to-sample reproducibility is a daily challenge that is also regularly cited as an area for improvement in size-selection.

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