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These application/technical notes are intended to help you to evaluate our products within a familiar context.
Our range of notes and articles are continually being updated and expanded.
Click on one of the numbered notes to view a synopsis and link to pdf. If you do not have Adobe Reader installed on your computer, you can download it free by clicking on this link:
1 Cooled CCD Camera Technology Terminology
Sensitivity and performance with any CCD digital imaging system can be significantly affected by several key technology specifications. When looking for a CCD imaging system with high sensitivity for fluorescence and other low light applications such as chemiluminescence, these critical technology features should be extensively evaluated for highest sensitivity, throughput, minimal exposure times, and image quality.
2 Blue Converter Screen For Molecular and Cellular Biology Applications
Fluorescent dyes and proteins are basic analytical tools used in many molecular and cellular biology applications. Fluorescent dyes such as ethidium bromide, SYBRr Green, SYBRr Gold and SYPROr Orange are used to detect nanogram quantities of DNA, RNA and protein separated by gel electrophoresis techniques.
3 Chemiluminescence vs Chemifluorescence
Traditionally, radio-isotopic protocols have always been standard in the laboratory. But with costs and concerns associated with using and disposing of radioisotopes escalating, many researchers are turning to non-isotopic methods wherever possible. Consequently, new chemistries are being developed to substitute radio-isotopic methods. The most popular method currently is that of chemiluminescence although some scientists prefer chemifluorescence. So how do these differ and which is better?
4 Digitisation and Data Dynamic Range
This application note has been written to provide a guide to the terminology used to define "dynamic range", and the important technical factors which CCD imaging systems require to generate accurate and linear scientific data.
5 Epi-illumination versus Transillumination
Syngene image capture systems offer the flexibility of using both transmitting and epi (reflective) visible and ultra violet (UV) light sources. Often confusion can arise as to which one is best for which application.
6 Visualising Green Fluorescent Protein
Green fluorescent protein (GFP) from Aequorea victoria (jellyfish) has stimulated a great deal of excitement among molecular, developmental and cell biologists. This is because GFP is a small protein (27 Kd) and the DNA sequences coding for GFP can be manipulated by recombinant DNA technology to create gene fusions between GFP and any protein of interest. Such DNA constructs can then be introduced into living cells to express the GFP fluorescent tag on the protein of interest to the investigator.
7 Colony Counting Using GeneTools
The colony counting software allows you to count objects such as bacterial colonies or plaques on a petri dish or multi-well plate. The software will automatically count colonies, separating clusters so there is no need for manual intervention. Counting within a user definable frame, the results are fast and accurate.
8 Neutral Fielding - for Syngene Image Capture Systems
Some of our competitors use a Flat Field Correction method in order to address uneven illumination of light sources. This involves subtracting the image information from an empty field of view or ‘perfectly flat’ fluorescent reference sample from that of the same field of view with the gel added. As this process involves the subtraction of one image from another, the integrity of the raw data of the initial captured image is compromised. Such manipulation is incompatible with Good Laboratory Practice (GLP) and therefore not adopted by Syngene. The Neutral Fielding Correction method utilised by Syngene addresses uneven light illumination whilst maintaining GLP compliance.
9 Glossary of CCD Terminology
10 Which Excitation Wavelength and which Emission Filter should I use?
Syngene provides full flexibility throughout the range of imaging systems, with excitation energy sources available for all fluorescent, colorimetric and chemifluorescent applications.
11 File Formats that can be used and saved in GeneSnap and GeneTools
GeneSnap stores images in a unique *.SGD file format. This format maintains the integrity of the original data and guarantees the accuracy of the analysis. Data stored and analysed using this format complies entirely with Good Laboratory Practice (GLP). These images are saved using the "Save Image As" command in the File menu.
12 Pixel Binning - Improved Sensitivity?
Pixel Binning is a process that further enhances sensitivity of a CCD sensor in terms of the speed of image acquisition. This process involves taking square groups of pixels and combining them into one `super' pixel, capable of holding much more light. This has the effect of reducing required exposure times. It is however important to realise that the drawback of pixel binning is a reduction in image resolution.
13 Multi-Layered Gel Analysis - For High Throughput Screening
Research often involves the use of multi-layers within gels. This enables high-throughput screening in order to maximise sample electrophoresis. In the past, analysis has had to be carried out by manipulating a grid around each layer and then repeating for each well layer, quite laborious and time consuming.
14 Multiplexing Gel Samples
It is becoming increasingly common within the life science laboratory to use more then one fluorescent label within the same gel. Sometimes up to three different stains will be used to identify different samples on the same gel. In order to image these stains, additional specific filters must be used.
15 Colourimetric markers
A common life science application using western blot membranes involves labelling standard lanes with a colorimetric marker, and unknown sample protein bands with a chemiluminescent substrate such as the GE Healthcare (Amersham) ECL+ Kit.
16 GeneTools MADGE - a New Tool for High Throughput Screening
MADGE, or Micro Array Diagonal Gel Electrophoresis as it is fully known, is a simple and highly effective method for high throughput screening. It involves using a 96 well gel in exactly the same format as a 96 well plate with electrophoresis occurring at an angle such that the elecrtophoretic path bypasses adjoining rows of wells. By running the gel using this diagonal path, a run length of up to 26.5mm can be achieved. This allows enough separation to calculate molecular weight data and identify doublet bands. As well as the 96 well format, 192 or even 364 wells can be run out simultaneously using MADGE.
17 On Chip Integration versus Image Additions in Series Capture
Series Capture is a function of GeneSnap Software that gives the user flexible options for image acquisition.
18 How to use Data from GeneTools
Many researchers today want to use the data obtained from analysing their images with Syngene image capture systems in presentations, grants, papers and posters. This often requires exporting data to a spreadsheet program for additional processing.
19 Using the UV safety over-ride facility
As an essential safety feature, the UV light is switched off automatically whenever the darkroom door is opened. However, on occasions, the user may wish the UV light to remain on in order that the gel can be viewed by eye or to cut out bands for further analysis.
20 What is Background Subtraction?
Baseline correction is used to reduce the effect of background and to emphasise the signal.
21 Visualising SYBR GreenTM Using a Syngene Image Capture System
SYBR GreenTM, from Invitrogen (Molecular Probes) is increasingly used as an alternative to Ethidium bromide in the visualisation of nucleic acids. SYBR GreenTM, chelates (binds) directly to the nucleic acid. Upon excitation with an appropriate light source, a green light is emitted that can be imaged and quantified.
22 Re-aligning Gel Tracks in GeneTools
When analysing a gel in GeneTools, the process has always been much easier when the gel tracks run vertically from top to bottom. This is because the software windows are much easier to work with and view when the data is displayed in this format.
23 How to Enhance your Image in GeneSnap
Enhancement and Annotation - All tools are present on one screen.
24 Using the Manual Grid Adjusters in GeneTools
Within GeneTools, the track manipulation tool bar exists in order to adjust the grid around your gel sample in order to get the most from the analysis.
25 For Colony and Cell Counting Applications GeneTools offers Invaluable Features
When counting colonies or cells using an image analysis system it is vitally important to choose software which is versatile enough to be able to quickly handle routine counting, yet is simple enough to provide as much automation as possible. To guide users, Syngene presents in this application note a selection of ways in which GeneTools, the software which comes as standard with Syngene image capture systems, can help with colony and cell counting. The array of GeneTools applications has been developed by Syngene from many years' experience of producing integrated imaging equipment as well as the consultation and feedback given by many of the international scientists using Syngene systems.
26 For Accurate Protein Gel and Blot Analysis, GeneTools is the Best Solution
Capturing an image of a protein gel or blot with an image analysis system is just the start of determining what information that image has to yield. As a guide to users, Syngene presents in this application note a selection of examples of the commonest uses of GeneTools with protein gels and blots. GeneTools is the software provided as standard with Syngene image capture systems. The array of GeneTools applications has been developed by Syngene from many years' experience of producing integrated imaging equipment as well as the consultation and feedback given by many of the international scientists using Syngene systems.
27 GeneTools - the Essential Software for Accurate DNA/RNA Gel and Blot Analysis
Capturing an image of a DNA/RNA gel or blot with an image analysis system is only the start of determining what information that image has to yield. To help guide users, Syngene presents in this application note a selection of DNA/RNA gel and blot applications of GeneTools, the software included with Syngene's image capture systems. The array of GeneTools applications has been developed by Syngene from many years' experience of producing integrated imaging equipment as well as consultation and feedback given by many international scientists using Syngene systems.
28 Imaging Chemiluminescent blots
With the availability of CCD camera systems suitable for chemiluminescence detection, such as G:BOX Chemi HR16, G:BOX Chemi XT16 and GeneGnome from Syngene, many scientists are now imaging their blots using these systems instead of film. There are many advantages in using a CCD camera for imaging: greater dynamic range, ease of analysis, no darkroom or messy developing solutions required, multiple images may be captured, image saturation can be automatically detected etc. To achieve the best results from a CCD camera, it is important to optimise capture conditions. This application note will guide the new user through the procedure, using an image capture system from Syngene.
29 How to Perform Internal Calibration with GeneTools
Syngene recommends internal rather than external calibration of each gel or autoradiograph to produce the most precise quantification results. The calibration must take into account all the conditions used at the time of the experiment. Any differences in the gel, temperature, speed or buffer need to be considered because they are likely to have an impact on how the DNA or protein bands appear on the gel.
30 GeneDirectory- A new tool for genetic fingerprint analysis
Molecular biology researchers of today are confronted with vast quantities of data, a sizeable proportion of which is genetic fingerprint information. This information is used for phylogenetic studies, forensic science, paternity studies and indeed any application where a genetic profile has to be compared with one, or several, others. Comparing gel band patterns was revolutionized by the advent of gel analysis packages such as GeneTools.
31 Recommended environment for Syngene Image Capture Systems
Recommended operating specifications are detailed.
32 Visualising SYBR GoldTM
SYBR GoldTM from Invitorgen (Molecular Probes) is a more sensitive fluorescent dye compared to ethidium bromide in the visualisation of nucleic acids. SYBR GoldTM binds directly to ss and ds RNA and DNA in electrophoretic gels. This stain penetrates thick gels easily for fast and even staining. Upon excitation with an appropriate light source, a green light is emitted that can be imaged and quantified.
33 A Comparison of UV to Blue Light Converters
UV to blue light converters and blue light excitation sources are becoming increasingly popular as although less sensitive than UV, some users prefer this option from a safety point of view: the gel may be viewed by eye without the need for safety precautions. In addition a converter can be easily positioned on a UV transilluminator in a gel documentation system and therefore does not necessitate the use of expensive lasers.
34 GeneGenius used for imaging fluorescently labelled riboprobes
In-situ hybridisation using riboprobes labelled with fluorescein is a commonly used technique for identifying specific mRNA involved in a range of human diseases and disorders. However, one difficulty with using riboprobes labelled in this way is finding a protocol that is sensitive enough to directly determine if they are sufficiently labelled before using them. One method of determining the amount of labelled riboprobe generated is to measure by standard agarose gel techniques the relative amount of RNA produced from the in-vitro transcription step. However, since this is only a measurement of the relative amount of RNA transcribed and not the incorporation of the labelled nucleotide it will provide a ‘quick and dirty’ estimate of the amount of label incorporated, which could be highly inaccurate.
35 G:BOX Chemi HR16 & XT16 for imaging ribonuclease protection assays
When using animal models for research into the pathology of different diseases, there is often a lack of starting material from which to isolate RNA. This difficulty arises because the most commonly used animal models (mice or rats) have small internal organs and as animals are expensive to purchase and maintain, the initial organ material is often also divided among many research groups. The knock on effect is that scientists frequently have a sample in the milligram range from which to isolate RNA, thus restricting the type of tests they can perform. Therefore, finding ways of maximising sensitivity is of extreme importance and as a result, one method that is gaining in popularity, is the Ribonuclease Protection Assay (RPA).
36 Illumination options available on Syngene image capture systems
Syngene now offers a wide range of darkroom options. All of these differ slightly in the lighting options that are fitted as standard, and those available as options. To avoid any confusion, this application note details which options are fitted or available with each system type.
37 Imaging Sypro™ Ruby and Deep Purple™ stained gels with G:BOX Chemi HR16 & XT16
Syngene’s G:BOX Chemi HR16/XT16 multi-functional image analysis system can be used to detect and analyse the sensitive protein stains, Deep Purple™ and Sypro™ Ruby. This will benefit users looking for an affordable, time saving method of automating their gel-based proteomics studies.
38 G:BOX Chemi HR16 for imaging large gold stained immunoblots allows rapid and safe analysis of 200 kD diseases associated proteins
Often researchers need to run longer protein gels to separate out and visualise proteins of 200 kD or larger. However, working with longer protein gels or the large Western blots produced using these gels can mean scientists have difficulty finding automated systems to accommodate these gels or blots. To overcome this problem some researchers label Western blots with a 125I secondary antibody and analyse them with an automated PhosphoImager. This is not ideal, as working with radioactive label poses a safety risk and the label requires stringent disposal methods. Additionally, a PhosphoImager and radioactive label are expensive purchases, which may not be within the budget of some laboratories.
39 NovaGlo Conversion Screen
Syngene now offers the NovaGlo conversion screen for the imaging of transmission samples (Silver gels, Coomassie Brilliant Blue (and other blue stains) gels and autoradiography film, in its newly designed darkroom, G:BOX.
40 Dyversity imaging of various fluorescent and visible protein dye labelled gels
The purpose of this report is to compare the dynamic range and linearity of a range of fluorescent and visible protein dyes. These being, Sypro Ruby® (Invitrogen), Deep PurpleTM (GE Healthcare), FlamingoTM (BioRad Laboratories), Coomassie Blue G-250 (GE Healthcare), Silver (Sigma) and the phosphoprotein dye ProQ DiamondTM (Invitrogen) using a variety of illumination options in the Dyversity imaging system
41 Dyversity and Typhoon 9400 imaging of Cy™ dye 1D and 2D gels
The purpose of this application note is to compare the dynamic range, linearity, sensitivity and image quality of the Dyversity imaging system with the Typhoon™ 9400 laser scanner (GE Healthcare).
42 G:BOX Chemi for rapid multiplex analysis
Multiplex analysis of proteins can be used to determine regulation of specific proteins, to diagnose disease pre-disposition or to accurately quantify protein amounts. Traditionally, multiplex analysis involves scientists producing and comparing a series of Western blots, a task, which is both time consuming and expensive in terms of multiple reagent and blot use. The ideal situation is to analyse a number of proteins on one Western blot, however, many detection methods such as colorimetric probes or chemiluminescence are unsuitable for multiplex analysis of different proteins on the same blot. This is because they produce only one colour or a white chemiluminescent emission so that users cannot distinguish between different proteins, especially when these proteins have the same molecular weight or are very close together.
43 Dyversity imaging of CyTM dye 1D and 2D gels
Assesing the performance of the Dyversity system for the imaging of Cy dye gels.
44 What is a 16 bit CCD camera?
Typically a 16 bit camera is one whose CCD is able to detect 65,536 grey levels. Put more simply this is 216 = 65,536. Since these cameras provide a lot more data than an 8, 10 or 12 bit camera they are therefore considered to offer superior advantage when working in quantitative modes.
45 Comparing the use of effective pixels with the native resolution of the camera
An autoradiograph film was imaged with a G:BOX Chemi HR16 at 1.38m pixels and 5.52m pixels. The native resolution of this camera is 1.38m pixels.
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