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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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

Better, Faster, Cheaper: Measuring the Speed of Science

Are we better off now than we were 10 years ago? Often times this question is answered subjectively and will vary from person to person. We can empirically show how life expectancy has increased over the centuries thanks to advances in the fields of agriculture and medicine, but what about quality of life? Science affects our lives every day, and the general notion is that better science will (eventually) translate into better lives. There is a burning curiosity shared by myself and others to quantify how we have progressed in science over the years:

publication-growth

Click for full article. Source: Bornmann, L. & Mutz, R. (2015). Growth rates of modern science: a bibliometric analysis based on the number of publications and cited references. Journal of the Association for Information Science and Technology, 66(11), 2215–2222.

Bornmann and Mutz demonstrate in the image shown above how we have been doubling scientific output every nine years since the 1940s. That is not to say that we have become twice as smart or efficient; this phenomenon could be partially fueled by a desire to gain prestige through a high number of publications. To better assess the topic of efficiency, we can measure how long it takes to perform specific procedures and how much they cost. This article compares the rate of improvement for DNA sequencing, PCR, GC-MS and general automation to the rate of improvement for supercomputers and video game consoles.

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T-Vector Cloning: Answers to Frequently Asked Questions

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

qPCR: The Very Basics

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. Data are presented graphically rather than as bands on a gel.

For those of us well versed in traditional, end-point PCR, wrapping our minds and methods around real-time or quantitative (qPCR) can be challenging. Here at Promega Connections, we are beginning a series of blogs designed to explain how qPCR works, things to consider when setting up and performing qPCR experiments, and what to look for in your results.

First, to get our bearings, let’s contrast traditional end-point PCR with qPCR.

End-Point PCR qPCR
Visualizes by agarose gel the amplified product AFTER it is produced (the end-point) Visualizes amplification as it happens (in real time) via a detection instrument
Does not precisely measure the starting DNA or RNA Allows you to measure how many copies of DNA or RNA you started with (quantitative = qPCR)
Less expensive; no special instruments required More expensive; requires special instrumentation
Basic molecular biology technique Requires slightly more technical prowess

 

Quantitative PCR (qPCR) can be used to answer the same experimental questions as traditional end-point PCR:  Detecting polymorphisms in DNA, amplifying low-abundance sequences for cloning or analysis, pathogen detection and others. However, the ability to observe amplification in real time and detect the number of copies in the starting material allow quantitation of gene expression, measurement of DNA damage, and quantitation of viral load in a sample and other applications.

Anytime that you are preforming a reaction where something is copied over and over in an exponential fashion—contaminants are just as likely to be copied as the desired input. Quantitative PCR is subject to the same contamination concerns as end-point PCR, but those concerns are magnified because the technique is so sensitive. Avoiding contamination is paramount for generating qPCR results that you can trust.

  1. Use aersol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps.
  2. Wear gloves. Change them frequently.
  3. Have designated areas for pre- and post-amplification work.
  4. Use “master mixes” to minimize variability. A master mix is a ready-to-use mixture of your reaction components (excluding primers and sample) that you create for multiple reactions—because you are pipetting larger volumes to make it, and all of your reactions are getting their components from the same master mix, you are reducing variability from reaction to reaction.
  5. Dispense your primers into aliquots to minimize freeze-thaw cycles and the opportunity to introduce contaminants into a primer stock.

 

These are very basic tips that are common to both end-point and qPCR, but if you get these right, you are off to a good start no matter what your experimental goals are.

Dealing with PCR Inhibitors

InhibitionThe polymerase chain reaction (PCR) has revolutionized modern biology as a quick and easy way to generate amazing amounts of genomic data. However, when PCR doesn’t work, it can be frustrating. At these times, PCR and reverse transcription PCR (RT-PCR) inhibitors seem to be everywhere: They lie dormant in your starting material and can co-purify with the template of interest, and they can be introduced during sample handling or reaction setup. The effects of these inhibitors can range from partial inhibition and underestimation of the target nucleic acid amount to complete amplification failure. What is a scientist to do?

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Top Ten Tips for Successful PCR

We decided to revisit a popular blog from our Promega Connections past for those of you in the amplification world. Enjoy:

magnesium-31

    • Modify reaction buffer composition to adjust pH and salt concentration.
    • Titrate the amount of DNA polymerase.
    • Add PCR enhancers such as BSA, betaine, DMSO, nonionic detergents, formamide or (NH4)2SO4.
    • Switch to hot-start PCR.
    • Optimize cycle number and cycling parameters, including denaturation and extension times.
    • Choose PCR primer sequences wisely.
    • Determine optimal DNA template quantity.
    • Clean up your DNA template to remove PCR inhibitors.
    • Determine the optimal annealing temperature of your PCR primer pair.

[Drum roll please]…and the  most important thing you can do to improve your PCR results is:

  • Titrate the magnesium concentration.

And if you want to, you can even build a custom PCR protocol using our iOS and Android device apps. Email it to your lab account, print it out for your notebook or just store it on your device for future reference.

DNA Purification, Quantitation and Analysis Explained

WebinarsYesterday I listened in on the Webinar “Getting the Most Out of Your DNA Analysis from Purification to Downstream Assays”, presented by Eric Vincent–a Product Manager in the Promega Genomics group.

This is the webinar for you if you have ever wondered about the relative advantages and disadvantages of the many methods available for DNA purification, quantitation and analysis, or if you are comparing options for low- to high-throughput DNA purification. Eric presents a clear analyses of each of the steps in a basic DNA workflow: Purification, Quantitation, Quality Determination, and Downstream Analysis, providing key considerations and detailing the potential limitations of the methods commonly used at each step.

The DNA purification method chosen has an affect on the quality and integrity of the DNA isolated, and can therefore affect performance in downstream assays. Accuracy of quantitation also affects success, and the various downstream assays themselves (such as end-point PCR, qPCR, and sequencing) each have different sensitivities to factors such as DNA yield, quality, and integrity, and the presence of inhibitors. Continue reading

PCR Protocols for Android

Over the years we have produced several articles and technical resources on PCR. One of the most widely used is a chapter on PCR in the Protocols and Applications guide, featuring an explanation of the techniques involved, tips for reaction optimization, and example protocols for routine PCR, qPCR and RT-PCR. The protocols and applications guide is part of the Promega App, available on the promega.com web site, and as an iPhone/iPad and Android App.

Recently, we released an update to the Android app that includes customizable PCR protocols. Right now, three protocols are available in this format (Basic PCR, Hot-Start PCR and qPCR). These custom protocols allow you to input specific values for your experiment, such as concentration of stock solutions, desired number of reactions, etc. These values are used to calculate the volumes required at each step in the procedure, and a customized protocol is generated with the volumes of each reagent calculated and incorporated. Once generated, protocols can be saved for re-use, or emailed to an account for subsequent printing or incorporation into a lab notebook. Continue reading

Troubleshooting T-Vector Cloning

Why do few of my pGEM®-T or pGEM®-T Easy Vector clones contain the PCR product of interest?

There are several possible reasons why the PCR product may not be recovered after ligation, bacterial transformation and plating when using the pGEM®-T or pGEM®-T Easy Vector Systems.

The PCR fragment may not be A-tailed. Without the A overhangs, the PCR product cannot be ligated into a T vector. Use a nonproofreading DNA polymerase like GoTaq® DNA Polymerase for PCR. If a proofreading DNA polymerase is used, A overhangs will need to be added. Purify the PCR fragment, and set up an A-tailing reaction (see the pGEM®-T and pGEM®-T Easy Vector Systems Technical Manual #TM042). The A-tailed product can be added directly to the ligation as described in the pGEM®-T or pGEM®-T Easy Vector protocol.

The insert:vector ratio may not be optimal. The ideal ratio for each insert to a vector can vary. For example, the Control Insert DNA works well at a 1:1 ratio, but another insert may be ligated more efficiently at a 3:1 ratio. Check the integrity and quantity of your PCR fragment by gel analysis. Optimize the insert:vector ratio (see Technical Manual #TM042).

Multiple PCR products were amplified and cloned into the pGEM®-T or pGEM®-T Easy Vector. Other amplification products including primer dimers will compete for ligation into the T vector, decreasing the possibility that the desired insert will be cloned. To minimize other competing products, gel purify the PCR fragment of interest.

Promega Technical Services Scientists are here to assist you in troubleshooting your experiments at any time. Contact Technical Services.