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:

Continue reading

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.

Continue reading

How Do I Choose the Right GoTaq® Product to Suit My Needs for EndPoint PCR?

We offer a wide array of GoTaq® DNA Polymerases, Buffers and Master Mixes, so we frequently answer questions about which product would best suit a researcher’s needs. On the product web page, you can filter the products by clicking the categories on the left hand side of the page to narrow down your search. Here are some guidelines to help you select the match that will best suit your PCR application. Continue reading

Six (and a Half) Reasons to Quantitate Your DNA

Knowing how much DNA you have is fundamental to successful experiments. Without a firm number in which you are confident, the DNA input for subsequent experiments can lead you astray. Below are six reasons why DNA samples should be quantitated.

6. Saving time by knowing what you have rather than repeating experiments. Without quantitating your DNA, how certain can you be that the same amount of DNA is consistently added? Always using the same volume for every experiment does not guarantee the same DNA amount goes into the assay. Continue reading

From Napkin Sketch to “Custom Kit”: CloneWeaver® Workflow Builder Gets Your Cloning Organized

20161018_150403Let’s face it, most lab techs and purchasing agents aren’t all that happy when you send them an Instagram picture of your latest lunchroom-napkin cloning strategy as your order form for your next big cloning experiment. So we have created the CloneWeaver® Workflow Builder. You can transfer your brilliance easily from that lunchroom napkin to an orderly email or print out of every vector, enzyme, purification kit, and transfection reagent your next big molecular cloning experiment requires. You can even save your one-of-a-kind “cloning kit” for future endeavors.

The CloneWeaver® tool will walk you through every step of the molecular cloning process from selecting a vector to finding a transfection reagent for mammalian cells. So if you are starting a new project, we are with you every step of the way. We will help you find restriction enzymes and even remind you about markers and biochemicals that you may want to have on hand for your experiment. Within the tool we have links to additional resources like our RE Tool and catalog pages if you need more help.

clone_weaverAlready have a favorite vector and a freezer full of restriction enzymes? No problem, skip those steps and move on to getting the perfectly sized nucleic acid markers or the particular polymerase your experiment requires.

Are you teaching a molecular genetics course? CloneWeaver® workflow builder is perfect for creating the list of laboratory reagents you are going to need for your students—and you will have this same list as a starting point for other lab experiments or classes later on because you can save the lists that you build. You can even pass them along to other professors.

So, if molecular cloning is in your future, let us help you get organized. Try the CloneWeaver® Workflow Builder.

Almost As Good As the Expert Down the Hall: The Citations Database

database_1When you are faced with a new research challenge or are troubleshooting in the lab, nothing replaces the wisdom of the lab tech down the hall who has 20 years experience doing the very technique you need to try.

But, sometimes there isn’t a local expert handy.

The Citations Database on the Promega website provides another source of expertise for you. We curate peer-reviewed publications that cite the use of Promega products so that you can see what people have done and how they have done it. We include links to PubMed for articles that are in indexed journals, and we also include brief notes about how the Promega products were used in the research.

Whether you are working with a new sample type, troubleshooting nucleic acid isolation, or trying a completely new assay, see how the Citations Database can help you in the laboratory. Continue reading

The Cell Line Identity Crisis: Old problems, new concerns

If you work with cell lines you may have paid attention to the dramatic headline published last month in the online journal STAT, Thousands of studies used the wrong cells, and journals are doing 29981164-Whistlenothing.” In their column The Watchdogs (“Keeping an eye on misconduct, fraud, and scientific integrity”), Ivan Oransky and Adam Marcus call out the fact that scientists continue to publish research using cell lines that are contaminated or misidentified. Recent estimates have found that the percentage of misidentified cell lines used by scientists is as high as 20 to 36. The blame here is being placed on the peer reviewed journals for not blowing the whistle. The authors call for journals to put some “kind of disclaimer on the thousands of studies affected.”

This is not a new claim. The continuing problem of cell line misidentification, of lack of authentication, has been covered before in various channels. It’s easy to find news publicizing yet another retracted publication. Promega Connections has published a number of blog posts addressing this, one as recently as last year: Do You #Authenticate? This post describes the bold move by the journal Nature to adopt a new policy around cell line authentication. Beginning in May 2015 the journal required authors of all submitted manuscripts to confirm the identity of cell lines used in their studies and provide details about the source and testing of their cell lines. Continue reading