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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.
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Hardware
Imaging
Software
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, radioisotopic 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
GFP is isolated from the Jellyfish Aequorea Victoria and is a member of a family of fluorescent proteins that have become widely used tools in many biological research applications.
7 Colony and cell counting using GeneTools software
GeneTools colony and cell counting software allows you to count bacterial colonies or plaques and cells present 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
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
Multi-layers within gels are often used in research for applications that require high throughput screening such as fingerprinting and genotyping. High-throughput screening enables sample electrophoresis to be maximised. In the past, analysis has had to be carried out by manipulating a grid around each layer and then repeating for each well layer, which can be quite laborious and time consuming.
14 Multiplexing Gel Samples
Multiplexing dyes is becoming increasingly common within the life science laboratory to use more than one fluorescent label within the same gel. Multiplexing is especially common for laboratories using Alexa Fluor and Qdot dyes (Invitrogen, UK). Often up to three different stains will be used to identify different samples on the same gel. In order to image these stains, additional specific emission filters must be used.
15 Colourimetric markers
Colorimetric markers are commonly used to assess the relative molecular weight (MW) of a protein on a gel; protein MW markers are normally run in the outer lanes of the gel for comparison.
16 GeneTools MADGE - a New Tool for High Throughput Screening
MADGE, or Micro Array Diagonal Gel Electrophoresis as it is fully known, is a method invented to serve the need of human molecular genetic epidemiology studies (Day and Humphries 1994: Analytical Biochemistry 222;389-95). MADGE is a simple and highly effective method for high throughput screening. It involves using a 96 well format gel with electrophoresis occurring in a 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. 192 and 364 wells format gels are compatible with MADGE and can run simultaneously.
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 dyes using a Syngene image capture system
SYBR dyes were introduced in 1993 and have become very popular nucleic acid gel stains due to their high sensitivity and ease of use. Commonly used SYBR dyes are SYBR Green, SYBR Gold and SYBR Safe.
22 Aligning banks of tracks in GeneTools for high throughput gels and E-gels
The E-Gel electrophoresis system is a system which does not require buffer for agarose gel electrophoresis of DNA samples. E-Gels are available in a variety of agarose percentages, well formats (48 and 96 well) and throughput capacities to suit specific applications.
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 GeneTools - the essential software for accurate DNA/RNA or protein gel and blot analysis
GeneTools software available with GeneSnap image capture software offers accurate. reproducible and quantitative analysis of numerous molecular biology based applications.
28 Imaging Chemiluminescent blots
With the availability of CCD camera systems suitable for chemiluminescence detection, such as G:BOX iChemi XR, G:BOX iChemi XT and GeneGnome from Syngene, and the development of chemiluminescence kits, many scientists are now imaging their blots using these systems instead of film.
29 How to Perform Internal Calibration with GeneTools
Internal calibration with GeneTools is recommended by Syngene instead of external calibration of each gel or autoradiograph to generate quantitative results every time. 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 as they may have an impact on how the DNA or protein bands appear on the gel.
30 GeneDirectory- A new tool for genetic fingerprint analysis
Phylogenetic studies often generate vast quantities of data, a sizeable proportion of which is genetic fingerprint information. This information is used for forensic science, paternity studies and indeed any application where a genetic profile has to be compared with one, or several, others. Gel band pattern comparisons were revolutionized by the arrival of gel analysis packages such as GeneTools. GeneTools allows a user to store the information associated with a gel in a GLP manner, perform bandmatching across a gel to a known standard and export this information. However, gel analysis packages do not allow a user to compare gel band patterns between gels. In the past if such analysis were required, it would be necessary for either the data to be exported to another package or to do the calculations by hand.
31 Recommended environment for Syngene Image Capture Systems
Recommended operating specifications are detailed.
35 G:BOX iChemi XR & XT for imaging ribonuclease protection assays
Gene expression and protein activity within a cell are of great interest to researchers. Changes in cellular mRNA levels are of particular interest as these changes directly correlate in their corresponding protein levels. There are, however, exceptions to the rule. Many scientists study gene expression by monitoring the response of mRNA molecules to genetic manipulations. This is often accomplished by using a number of different techniques including Northern blotting, RT-PCR and nuclease protection assays.
36 Illumination options available on Syngene image capture systems
Syngene offers a variety of image capture systems to meet all your imaging requirements. There are several lighting options including transilluminator UV (trans UV), transilluminator white/visible light (trans white/ visible), transilluminator blue (trans blue) and Epi RGB, UV and white available which can be fitted as standard or purchased as an optional extra.
37 Imaging Sypro™ Ruby and Deep Purple™ stained gels with G:BOX iChemi XR & XT
SYPRO Ruby is a protein stain for the detection of proteins separated by polyacrylamide gel electrophoresis (PAGE). SYPRO Ruby stain can be used with either 1D or 2D gels. The SYPRO Ruby stain has equivalent to or better sensitivity than silver staining techniques.
39 NovaGlo Conversion Screen
Syngene offers the NovaGlo conversion screen for imaging of transmission samples such as silver stained gels, coomassie brilliant blue (and other blue stains) gels and autoradiography film.
41 Dyversity and Typhoon 9400 imaging of Cy™ dye 1D and 2D gels
To compare the dynamic range, linearity, sensitivity and image quality of the Dyversity imaging system (Syngene UK) with the Typhoon(TM) 9400 laser scanner (GE Healthcare, UK).
42 G:BOX iChemi 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.
47 Cy2, Cy3, Cy5 1D gel imaging using Cy dye lighting unit and epi RGB LED module
Comparing the epi RGB LED module with the Dyversity Cy dye lighting unit in terms of specificity, sensitivity and dynamic range.
48 Imaging Ethidium bromide dye
Recommended lighting and filter selection for imaging Ethidium bromide dye
49 Imaging Alexa Fluor 647 dye
Recommended lighting and filter selection for imaging Alexa Fluor 647 dye
50 Imaging Quantum dot (Q-dot 565 and 705) nanocrystals
Recommended lighting and filter selection for imaging Q-dot 565 and 705 nanocrystals
51 Imaging SYBR Safe gel stain
Recommended lighting and filter selection for imaging SYBR Safe gel stain
52 Imaging SafeView
Recommended lighting and filter selection for imaging SafeView
53 Imaging Cyanine 3
Recommended lighting and filter selection for imaging Cy3
54 G:BOX iChemi image analyzer and KPL chemiluminescent substrates make detecting proteins in picogram and femtogram range quick and easy
Western blotting is one of the most commonly used applications in protein research. Recent advances in chemiluminescent substrate design have meant when chemiluminescent blots are scanned using CCD image analysis systems, they can generate real-time images, in which smaller amounts of protein can be detected than on blots labeled with traditional colorimetric or fluorescent dyes.
55 G:BOX iChemi DyLight conjugates for imaging fluorescent Western blots - optimum lighting and filter combinations for maximum sensitivity
Recent advances in fluorescent dye technology allow for their use in Western blotting, which offers a number of benefits.
56 Imaging protein gels using GeneSnap software
Protein gels are routinely run in many life science laboratories and with an increasing number of gel stains available for staining proteins after electrophoresis it can be confusing as to which lighting and filter selection are required when imaging your protein gel.
57 Quantifying expression standards and expression values using GeneTools software
To analyse western blots in a quantitative manner a “standard” such as an internal control or an external standard such as the purified protein of interest in a known concentration (enables absolute quantification) are required.
59 Multiplexing LI-COR Infra red 680 and 800 dyes
Life sciences research has recently seen a trend towards the application of fluorescence-based detection of proteins on Western blots. Using fluorescence based fluorophores increases sample throughput and can make multiplexing several fluorophores less time consuming.
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