Welcome to the BIOKÉ NEWS FLOW! The BIOKÉ NEWS FLOW is published 3 times a year dedicated to providing you with the most recent application- and product updates, an interview and our current special offers.

The NucleoSpin® RNA Blood procedure is a simple and convenient method to isolate RNA from fresh or frozen whole blood stabilized, for example, with EDTA, citrate, or heparin.

The simple and efficient direct total blood lysis allows RNA isolation from whole blood without the common but cumbersome selective erythrocyte lysis at 4°C. Thus the complete procedure is performed at room temperature. Blood cells are lysed in Lysis Buffer DL. The RNA is bound to the NucleoSpin® RNA Blood Binding Column. DNA is efficiently removed by an on-column rDNase digestion. Contaminants and inhibitors are removed with two different wash buffers. Finally RNA is eluted with RNase-free water.

Higher yield from smaller sample volume - comparison to competitor kits RNA was isolated from the indicated blood volumes following each manufacturer's protocol. The quality of isolated RNA is comparable. Average RIN numbers (data not shown) were: 7.5 (Q); 7.7 (NucleoSpin® RNA Blood); 8.8 (NucleoSpin® RNA Blood Midi); 8.9 (Q, PAXgene®).

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* BIOKÉ Supplies MACHEREY-NAGEL in The Netherlands only.

Thermal expansion of Polypropylene (PP) plates leads to evaporation from outer wells.

PP is the optimum material for PCR tubes. It provides the most efficient heat transfer, as well as an inert surface with low binding capabilities for nucleic acids, proteins and other molecules. However, the material is not thermally stable in a plate format and expands and contracts during each PCR cycle (Figure 1). Such thermal expansion will weaken the plate seal and leads to sample evaporation mainly from corner wells and outer rows.

Figure 1: Standard plates with polypropylene frame expand by up to 2mm during thermal cycling which leads to movement of wells away from the plate centre. This movement is most significant in corner positions and outer rows of the plate. Sealing sheets do not expand at this rate so that the movement of wells will weaken the seal and lead to evaporation especially in corner positions and outer rows.

Thermal cycler Blocks do not prevent thermal expansion of PCR plates.

PCR blocks do not support PCR plates from the sides and the high temperatures from the thermal block and heated lid accelerate expansion of the plates (Figure 2).

Figure 2: Side-on view of a PCR plate in a thermal cycler. The sealed plate is sandwiched between the cycler block and the heated lid but it is only partly fixed in position at the bottom of tubes, allowing the plate to expand horizontally.

FrameStar® 2-component technology reduces thermal expansion and sample evaporation

The polycarbonate frame of FrameStar® plates is more heat resistant than standard PP plates which reduces thermal expansion to a minimum. For this reason the seal integrity remains intact even at elevated temperatures during PCR.

To illustrate this advantage of our two-component technology we have compared evaporation from one piece PP plates and FrameStar® PCR plates: Each well of a non-skirted 96well plate (single piece, PP) and a Framestar® non-skirted design (code 4ti-0710) was filled with 10μl H2O. The plates were sealed with a qPCR adhesive (code 4ti-0560) and the weight of plates was measured before and after performing PCR (30 cycles x 15" 95°C; 15" 55°C).

Table 1 shows that the average volume loss from one piece PP plates was 2.3µl per well which is equal to 23% of the total reaction volume. In contrast the volume loss from FrameStar plates was only 0.49µl per well using adhesive sealing.

Plate type	Starting Weight	Weight Post PCR	Weightloss	Volume loss total/per well
Framestar 4ti-0710	26,678g	26,631g	26,631g	47µl/0.49µl
One piece	17,807g	17,586g	0,221g	221µll/2.3µl

Table 1: Weight and volume loss from 96well PCR plates. Results shown are averages from 5 plates of each plate type. One piece PP plates showed more than 4 times higher volume loss than FrameStar® plates.

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The NEW SimpleChIP® Plus Chromatin IP Kits from Cell Signaling Technology are optimized to detect endogenous levels of protein-DNA interactions in both cultured cells and tissue samples.

These kits contain all reagents necessary to perform enzymatic digestion-based chromatin immunoprecipitation (ChIP) experiments quickly and easily from tissue samples, as well as positive and negative controls that ensure confidence in your results. The kits are available with either Protein G agarose (#9004) or Protein G (#9005) magnetic beads and contain all buffers and reagents needed to perform up to 30 ChIP assays.

Quick and easy scaling

A complete assay can be performed in as little as two days and can easily be scaled up or down for use with more or less cells or tissue sample. Enzymatic fragmentation of chromatin is much milder than sonication and eliminates problems resulting from variability in sonication power and emulsification of chromatin during sonication, which can result in incomplete fragmentation of chromatin or loss of antibody epitopes due to protein denaturation and degradation.

The SimpleChIP® Plus Enzymatic Chromatin IP Kit can be utilized with any ChIP-validated antibody to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells and tissue samples. Read more »

Figure: Mouse brain and mouse liver tissue were prepared and disaggregated into a single-cell suspension using a Dounce homogenizer and tissue disaggregator, respectively. The cells were then formaldehyde-crosslinked and chromatin was prepared and digested. DNA was purified and 10 μl was separated by electrophoresis on a 1% agarose gel and stained with ethidium bromide. The majority of chromatin from both brain (lane 1) and liver (lane 2) was digested to 1 to 5 nucleosomes in length (150 to 900 bp).

More information about these new SimpleChip® kits »

BIOKÉ offers a wide range of products for Next Generation Sequencing, from DNA purification* to NGS Data Analysis. Recently new Sample & Library Preparation products from NEB are launched that are compatible with the Ion PGM™ sequencer.

DNA / RNA Purification

MACHEREY-NAGEL* offers ready-to-use DNA and RNA purification kits in various formats including high-throughput applications to get your DNA or RNA. Read More »

Single Cell Amplification (optional)

The Single Cell WGA Kit from New England Biolabs efficiently amplifies total DNA from single cells or their DNA equivalent about 1 million-fold to produce 2–5 micrograms of amplified DNA in less than 3 hours. The kit also works successfully over a range of inputs up to thousands of cells or several ng of DNA. Read More »

Sample & Library Preparation

NEBNext® reagents from New England BioLabs are a series of highly pure reagents that facilitate sample preparation of DNA or RNA for next generation sequencing.

Available in sets, master mixes and modules, these robust reagents undergo stringent quality controls - additional QCs ensure maximum quality and purity - and functional validation. Besides the reagents for the Illumina, 454 and SOLiD platforms, NEBNext reagents are now also compatible for sequencing on a Ion Torrent™ PGM™.

Now compatible with Ion PGM™ sequencer

The NEBNext® Fast DNA Fragmentation & Library Prep Set 4 contains enzymes and buffers in convenient formats that are ideally suited for sample preparation for sequencing on an Ion PGM™ sequencer. Read More »

The NEBNext® Fast DNA Library Prep Set 4 contains enzymes and buffers in convenient formats that are ideally suited for sample preparation for next-generation sequencing on the Ion PGM™ sequencer. Read More »

All NEBNext reagents must pass rigorous quality control standards and are lot controlled, both individually and as a set of reagents.

Available soon: NEBNext Multiplex Oligos for Illumina (Index Primers 1–12) contains adaptors and primers that are ideally suited for multiplex sample preparation for next-generation sequencing on the Illumina platform.

More information about the NEBNext® kits

Sequencing

BIOKÉ offers a range of products from DNA purification to NGS Data Analysis. For sequencing instrument details, contact the relevant suppliers.

Data analysis

NextGENe® Software is a complete analysis package designed for use by biologists in the analysis of data from Next Generation Sequencing systems. The easy-to-use Windows® interface significantly reduces bioinformatic steps, provides annotated analysis review, while reducing sequencing errors to improve analysis accuracy and speed. Read More »

Open Application Note "Processing IonTorrent Sequencing Data using NextGENe® Software"
Open Application Note "Processing Ion AmpliSeq™ Cancer Panel Data using NextGENe® Software"
Open Application Note "Assembly of Bacterial Genomes from Ion PGM™ using the Floton™ Assembler in NextGENe® Software"

* BIOKÉ supplies MACHEREY-NAGEL products in The Netherlands only.

454™ is a trademark of Roche. SOLiD™ is a trademark of Life Technologies. Illumina® is a registered trademark of Illumina. PGM™ Torrent is a trademark owned by Life Technologies, Inc.

The Access Array System is the first high-throughput, target-enrichment system designed to work with all of the major next-generation sequencing instruments. It enables the user to enrich hundreds of unique targets (such as exons) from a large number of samples, all at one time. The system combines the cost and throughput benefits of microfluidics with the proven performance and flexibility of PCR. Each microfluidic reaction chamber can accommodate up to 10X independent amplicons (multiplex), enabling the enrichment of up to 480 amplicons across 48 samples in a single run.

Four-primer amplicon tagging

While the Access Array System can be used for nearly any PCR-based enrichment strategy, four-primer amplicon tagging with Fluidigm protocol and reagents is an ideal method for creating amplicon libraries for any next-generation sequencing platform. In the four-primer amplicon tagging, the inclusion of a short Consensus Sequence (CS) Tag onto the 5' end of the target-specific primer and the three-prime end of the sequencing adaptor allows the user to design and validate only target-specific primers regardless of the sequencer type or level of sample multiplexing required. The barcode oligos with CS linkers are universal reagents in the four-primer amplicon tagging protocol and can be used with any target-specific primer sequences, greatly reducing the number of unique oligos that are required to conduct multiple targeted resequencing experiments.

Four-primer amplicon tagging

A 96 Access Array Barcode Primer Plate for the 454™ Titanium Sequencer and a bidirectional 384 Barcode Kit for Illumina® sequencers. This allows sequencing of 96 samples and 384 samples for 454™ and Illumina® sequencing, respectively, in a single sequencing run.

Easy workflow

An entire target enrichment experiment from genomic DNA to a finished amplicon library can be carried out with only five hands-on steps, and completed in four hours with minimal hands-on time.

Contact BIOKÉ at info@bioke.com for more information about the Access Array

454™ is a trademark of Roche. Illumina® is a registered trademark of Illumina.

Ensuring easy ordering

Are you still ordering by (handwritten) fax, email, phone or even by post? This is a time-consuming activity and relatively sensitive to errors. Wouldn't you be interested in ordering online? This will allow you to order more convenient and gives you the following benefits:

• View your personal pricing
• Save a wishlist
• Efficient re-ordering
• Just order in 4 simple steps:
1. Log In
2. Choose your product(s)
3. Add remarks
4. Place your order

Click here to register and experience the ordering process yourself!

Free delivery on online orders! In the second quarter of 2012 you will not pay any shipping & handling costs when ordering via our webshop. Saving money cannot be any easier!

If you have any special wishes or suggestions for our webshop, please contact us at info@bioke.com. With your help and knowledge, we can enhance our webshop service quality.

The Utrecht Centre for Tick-borne Diseases (UCTD) is focused on ticks and tick-borne diseases, which adversely affect animal and human health. The UCTD-group is headed by Prof. Dr. Frans Jongejan. One of the technicians is Michiel Wijnveld who is managing the lab, mentoring students, purchasing materials, and performing research. Projects Currently the group is working on eight different projects and one of those is the "Tick busters project" that started in 2005. For this initiative, vets and individuals in the Netherlands started collecting ticks and send these to Utrecht. It was found that several dogs were infected with a tick-born disease that normally doesn't appear in the Netherlands. This was the reason to prolong this project. Now, the group receives around 5.000 ticks per year that hosted on dogs, cats, hedgehogs, and other animals. Of these ticks, the species is determined, DNA extracted and presence of pathogens is checked. This results in an overview of the fluctuation of tick-species and pathogens in the Netherlands. The group is also developing a quick diagnostic test for vets to test for pathogens in the blood of the host. For this test, the Loop-Mediated Isothermal PCR (LAMP) technique will be used to amplify DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four designed primers that recognize a total of six distinct sequences on the target DNA. The DNA polymerase used, is the Bst DNA Polymerase from New England Biolabs. An inner primer containing sequences for both the sense and antisense strands of the target DNA initiates LAMP. The final products are stem–loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing alternately inverted repeats of the target in the same strand. These products can be seen with the naked eye. In this way, vets can perform these tests themselves in a small lab with a PCR machine only and there is no need for any additional instruments to determine if a certain pathogen is present in the tick or not. The group has a close collaboration with Pretoria University in South Africa. Prof. Dr. Jongejan is professor at that university as well. The people in Africa are more dependent of their cattle than here in the Netherlands. The pathogens that appear in Africa result in a high mortality rate of the cattle or a very low productivity, while the pathogens and risks for pathogen transmission in Europe is considered slightly less of a threat. In addition to the research projects, the group is also organizing a 2-week summer school course. This course is meant for advanced MSc students with the ambition to embark on PhD research related to ticks and tick-borne diseases and for PhD students to support their ongoing research on ticks and tick-borne diseases. The students will do a field trip and screen their own ticks. Read More » Tick Screening To screen the pathogens in the ticks, Michiel first checks if a pathogen is present from a certain family. This is done by performing one of four PCRs with general primers. To determine the specific pathogen in those families, Michiel is using reverse line blot hybridization. With this technique multiple samples can be analyzed against multiple probes to enable simultaneous detection of 40 different pathogens. The detection is based on chemiluminescence. If a pathogen is found from a certain family, but the specific pathogen cannot be determined by reverse line blot hybridization, the DNA is sequenced to determine the specific pathogen. In order to prepair a sample for sequencing, Phusion® DNA Polymerase from New England Biolabs is used to ensure high quality results. To extract DNA from ticks, Michiel is using the NucleoSpin® Tissue kit from MACHEREY-NAGEL. Before starting the included extraction protocol, he first blends the tick to ensure the lysis will be successful. They have tested other methods to damage the tick, like liquid nitrogen treatment and beads, but simply blending works the best. Another kit Michiel is using from MACHEREY-NAGEL is the NucleoSpin® Blood kit. This kit is used to isolate DNA from the blood of a host to test for pathogens. Michiel prefers these MACHEREY-NAGEL kits because of the good price-quality ratio. "The kits that we used to use were more expensive and the kits from MACHEREY-NAGEL work perfectly." The second reason to choose for these kits is the relation with BIOKÉ, the distributor of the kits: "It is a small company, and I like the ease of contacting them with questions about our experiments. I always receive a quick and clear answer." Future The awareness of tick-borne diseases is increasing, but still people are not aware of all the risks that can result after a tick bite in the Netherlands for animals and humans. The well-know pathogen is Borrelia that causes lyme disease, but the number of pathogens is growing in the Netherlands. Ticks are imported in all kind of ways: cattle are transported to other countries, dog-owners take their dog to exotic places and don't check for ticks. Some of these imported ticks can survive pretty easy in our homes and replicate fast. The trend is that more exotic pathogens are found in animals and humans that haven't been abroad at all. More research is needed for the tick-borne diseases. How do ticks transfer the pathogen to the host and how we can prevent disease transmission? More than enough challenges! NucleoSpin® is a registered trademark of MACHEREY-NAGEL. Phusion® is registered trademark and property of Thermo Fisher Scientific. Phusion® DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is now manufactured by New England Biolabs, Inc. under agreement with, and under the performance specifications of Thermo Fisher Scientific.

Michiel Wijnveld studied Biology and Medical Laboratory Research and performed his final internship at the Utrecht Centre for Tick-borne Diseases. He started as a research technician one year ago and in addition to his research projects he is labmanager, mentor of the students and general purchaser of the lab.

MACHEREY-NAGEL offers a wide range of RNA-kits to get RNA out of all your samples. Using the MACHEREY-NAGEL RNA-kits means saving money, even up to 50%! All the reagents, like DNase, and the plastics are included in the kit already. There is no need to buy extra reagents and plastics, in contrary to competitor kits.

Besides saving money, save also on your precious R&D time. Benefit from the highest quality & consistent yield: avoid repeat purifications and failed experiments.

MACHEREY-NAGEL offers a full suite of products for all your RNA extraction needs. You can purify RNA from a range of sample sizes and a wide variety of sample types.

Save even more money now!

Take advantage of 20% discount until March 31, 2012! This discount is valid for all RNA-kits, also for the newly introduced RNA Blood kits. Don't forget to mention the code "RNA-discount12" on your order.
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Photograph your Duck

With the MACHEREY-NAGEL deliveries in February and March 2012, you receive a Dutch MN Duck. Photograph your duck in the lab and email the photo to info@bioke.com. The funniest photo will be rewarded with a box with Dutch snacks.

* BIOKÉ supplies MACHEREY-NAGEL products in The Netherlands only

Cell Signaling Technology assembled different pathway posters to provide a brief and current overview of selected signaling pathways. You can request your favorite pathway poster now via BIOKÉ.

» Request your Pathway Poster

BIOKÉ will have a booth at location 2-C28 at Laborama 2012. We will present new products from our suppliers and will have promotions that are only valid for visitors of the Laborama. Don't miss this event!

Laborama 2012 will take place in Brussels Kart Expo in Groot-Bijgaarden on March 22 and 23, 2012.