Nucleic Acid Analysis

Six (and a Half) Reasons to Quantitate Your DNA

Posted on by Sara Klink

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 you should quantitate your DNA.

6. Saving time by knowing what you have rather than repeating experiments. If you don’t quantitate 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. Each time there is a new purified DNA sample, the chances that you have the same quantity as before are lessened. Consequently, without knowing the DNA concentration of the sample you are using, the amount of input DNA cannot be guaranteed and experiments may have to be repeated.

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

Posted on by Michele Arduengo

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.

Of Elephant Research and Wildlife Crime – Molecular Tools that Matter

Posted on by Nicole Sandler

Here at Promega we receive some interesting requests…

Take the case of Virginia Riddle Pearson, elephant scientist. Three years ago we received an email from Pearson requesting a donation of GoTaq G2 Taq polymerase to take with her to Africa for her field work on elephant herpesvirus. Working out of her portable field lab (a tent) in South Africa and Botswana, she needed a polymerase she could count on to perform reliably after being transported for several days (on her lap) at room temperature. Through the joint effort of her regional sales representative in New Jersey/Pennsylvania (Pearson’s lab was based out of Princeton University at the time) and our Genomics product marketing team, she received the G2 Taq she needed to take to Africa. There she was able to conduct her experiments, leading to productive results and the opportunity to continue pursuing her work.

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Easy Automated Genomic DNA Isolation for GMO Testing: From Vision to Reality

Posted on by Kelly Grooms
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The European Union (EU) has a zero tolerance policy for products containing any material from non-authorized genetically modified (GM) crops. Seed entering EU markets may not contain even trace amounts of non-authorized genetically modified material. In 2012, as the global use of GM crops increased, seed testing loads in the EU continued to build. Isolating genomic DNA (gDNA) using traditional manual methods was becoming impractical in the face of increasing amounts of material that required testing. There was a growing need for an automated method to isolate gDNA from seed samples. Working to address this need, a group of scientists from the Bavarian Health and Food Safety Authority collaborated with scientists from Promega Corporation to evaluate the Maxwell® 16 Instrument and the associated chemistry as possible a solution for the testing labs.

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A Crash Course in CRISPR

Posted on by Darcia Schweitzer

CRISPR is a hot topic right now, and rightly so—it is revolutionizing research that relies on editing genes. But what exactly is CRISPR? How does it work? Why is everyone so interested in using it? Today’s blog is a beginner’s guide on how CRISPR works with an overview of some new applications of this technology for those familiar with CRISPR.

Introduction to CRISPR/Cas9

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were discovered in 1987, but it took 30 years before scientists identified their function. CRISPRs are a special kind of repeating DNA sequence that bacteria have as part of their “immune” system against invading nucleic acids from viruses and other bacteria. Over time, the genetic material from these invaders can be incorporated into the bacterial genome as a CRISPR and used to target specific sequences found in foreign genomes.

CRISPRs are part of a system within a bacterium that requires a nuclease (e.g. Cas9), a single guide RNA (sgRNA) and a tracrRNA. The tracrRNA recruits Cas9, while sgRNA binds to Cas9 and guides it to the corresponding DNA sequence of the invading genome. Cas9 then cuts the DNA, creating a double-stranded break that disables its function. Bacteria use a Protospacer Adjacent Motif, or PAM, sequence near the target sequence to distinguish between self and non-self and protect their own DNA.

While this system is an effective method of protection for bacteria, CRISPR/Cas9 has been manipulated in order to perform gene editing in a lab (click here for a video about CRISPR). First, the tracrRNA and sgRNA are combined into a single molecule. Then the sequence of the guide portion of this RNA is changed to match the target sequence. Using this engineered sgRNA along with Cas9 will result in a double-stranded break (DSB) in the target DNA sequence, provided the target sequence is adjacent to a compatible PAM sequence.

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The Making of a Promega Product: Teamwork = Success for the Maxwell RSC® ccfDNA Plasma Kit

Posted on by Nicole Sandler

Ever think about the kinds of challenges R&D scientists run up against in the course of developing a new product? The development of the Maxwell® RSC ccfDNA (circulating cell-free DNA) Plasma Kit is a particularly interesting example. Its path to commercialization was characterized by a number of unexpected technical hurdles, yet each was overcome through creative troubleshooting and aided by valuable collaborations across departments. All had a hand in finally launching the kit last August.

26062525-ccfDNA_Mar25-blog-DNAThe product’s launch was an exciting milestone for Promega as research interest in the role of ccfDNA as biomarkers in human disease continues to grow. Elevated levels of ccfDNA have now been reported in patients with cancer, inflammatory disease, infections and cardiovascular disease. In pregnant women, up to 10% of ccfDNA can be attributed to the fetus, so critical fetal DNA analysis can now be conducted through maternal blood samples. There are many advantages in the ability to isolate and analyze ccfDNA, so the development of a kit with high throughput capability was a priority for the Nucleic Acid Purification R&D team. Continue reading “The Making of a Promega Product: Teamwork = Success for the Maxwell RSC® ccfDNA Plasma Kit”

T-Vector Cloning: Answers to Frequently Asked Questions

Posted on by Radhika Ganeshan
Blue/White colony screening helps you pick only the colonies that have your insert.
Blue/White colony screening helps you pick only the colonies that have your insert.

Q: Can PCR products generated with GoTaq® DNA Polymerase be used to for T- vector cloning?

A: Yes. GoTaq® DNA Polymerase is a robust formulation of unmodified Taq Polymerase. GoTaq®DNA Polymerase lacks 3’ →5’ exonuclease activity (proof reading) and also displays non-template–dependent terminal transferase activity that adds a 3′ deoxyadenosine (dA) to product ends. As a result, PCR products amplified using GoTaq® DNA Polymerase will contain A-overhangs which makes it suitable for T-vector cloning.

We have successfully cloned PCR products generated using GoTaq® and GoTaq® Flexi DNA Polymerases into the pGEM®-T (Cat.# A3600), pGEM®-T Easy (Cat.# A1360) and pTARGET™ (Cat.# A1410) Vectors.

Q: Can GoTaq® Long PCR Master Mix be used for T-Vector Cloning?

A: Yes it can. GoTaq® Long PCR Master Mix utilizes recombinant Taq DNA polymerase as well as a small amount of a recombinant proofreading DNA polymerase. This 3´→5´ exonuclease activity (proof reading) enables amplification of long targets. Despite the presence of a small amount of 3´→5´ exonuclease activity, the GoTaq® Long PCR Master Mix generates PCR products that can be successfully ligated into the pGEM®-T Easy Vector System.

We have demonstrated that GoTaq® Long PCR Master Mix successfully generated DNA fragments that could be ligated into pGEM®-T Easy Vector System without an A-tailing procedure, and with ligation efficiencies similar to those observed with the GoTaq® Green Master Mix.

For details refer to Truman, A., Hook, B. and Wieczorek, D. Using GoTaq® Long PCR Master Mix for T-Vector Cloning.

Tip: For cloning blunt-ended PCR fragments into T-vectors, use the A-tailing protocol discussed in the pGEM®-T and pGEM®-T Easy Technical Manual #TM042.

Q: How do I prepare PCR products for ligation? What products can be used to purify the DNA?

Continue reading “T-Vector Cloning: Answers to Frequently Asked Questions”

Top 10 Tips to Improve Your qPCR or RT-qPCR Assays

Posted on by Kelly Grooms
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Scientists have had a love-hate relationship with PCR amplification for decades. Real-time or quantitative PCR (qPCR) can be an amazingly powerful tool, but just like traditional PCR, it can be quite frustrating. There are several parameters that can influence the success of your PCR assay. We’ve highlighted ten things to consider when trying to improve your qPCR results.

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Real-Time (quantitative) qPCR for Quantitating Library Prep before NGS

Posted on by Promega

Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction.
Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction.

This the last in a series of four blogs about Quantitation for NGS is written by guest blogger Adam Blatter, Product Specialist in Integrated Solutions at Promega.

When it comes to nucleic acid quantitation, real-time or quantitative (qPCR) is considered the gold standard because of its unmatched performance in senstivity, specificity and accuracy. qPCR relies on thermal cycling, consisting of repeated cycles of heating an cooling for DNA melting and enzyamtic replication. Detection instrumentation is capable of measuring the accumulation of DNA product after each round of amplification in real time.

Because PCR amplifies specific regions of DNA, the method is highly sensitive, specific to DNA, and it can determine whether a sample is truly able to be amplified. Degraded DNA or free nucleotides, which might otherwise skew your quantiation, will not contribute to the signal, and your measurement will be more accurate.

However, while qPCR does provide technical advantages, the method requires special instrumentation, specialized reagents and is a more time-consuming process. In addition, you will probably need to optimize your qPCR assay for each of your targets to achieve your desired results.

Because of the added complexity and cost, qPCR is a technique suited for post-library quantitation when you need to know the exact amount of amplifiable, adapter-ligated DNA.  PCR is the only method capable of specifically targeting these library constructs over other DNA that may be present. This specificity is important because accurate normalization is especially critical for producing even coverage in multiplex experiments where equimolar amounts of several libraries are added to a pooled sample. This normalization process is essential  if your are screening for rare variants that might be lost in background and go undetected if underrepresented in a mixed pool.

 

Read Part 1: When Every Step Counts: Quantitation for NGS

Read Part 2: Nucleic Acid Quantitation by UV Absorbance: Not for NGS

Read Part 3: Fluorescence Dye-Based Quantitation: Sensitive and Specific for NGS Applications

Fluorescence Dye-Based Quantitation: Sensitive and Specific for NGS Applications

Posted on by Promega

This is the third post in a series of blogs on quantitation for NGS applications written by guest blogger Adam Blatter, Product Specialist in Integrated Solutions at Promega.

Fluorescent dye-based quantitation uses specially designed DNA binding compounds that intercalate only with double stranded DNA molecules. When excited by a specific wavelength of light, only dye in the DNA-bound state will fluoresce. These aspects of the technique contribute to low background signal, and therefore the ability to accurately and specifically detect very low quantities of DNA in solution, even the nanogram quantities used in NGS applications.

For commercial NGS systems, such as the Nextera Rapid Capture Enrichment Protocol by Illumina, this specificity and sensitivity of quantitation are critical. The Nextera protocol is optimized for 50ng of total genomic DNA. A higher mass input of genomic DNA can result in incomplete tagmentation, and larger insert sizes, which can adversely affect enrichment. A lower mass input of genomic DNA or low-quality DNA can generate smaller than expected inserts, which can be lost during subsequent cleanup steps, giving lower diversity of inserts.

Continue reading “Fluorescence Dye-Based Quantitation: Sensitive and Specific for NGS Applications”