FAQs
What concentration of template is required?
All samples must be submitted with accurate quantitations of template and primer. The primer must be at least 10pmol/µl. Also, all templates must be diluted to the correct concentration, falling within the following ranges:
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Plasmid preparation
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PCR product
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100 - 200 bp, 1 ng/µl - 3 ng/µl
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200 - 500 bp, 3 ng/µl -10 ng/µl
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500 - 1000bp, 5 ng/µl - 20 ng/µl
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1000 - 2000 bp, 10 ng/µl - 40 ng/µl
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> 2000 bp, 40 ng/µl -100 ng/µl
The upper detection limit of the CCD in the 3130xl genetic analyser is relatively low and thus easily swamped with excess signal. Therefore, please note that the template concentration must not exceed our recommended values or you will end up with a poor quality result. Comprehensive guidance notes are available to help you get the most out of the service.
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What volume of template and primer should I provide per reaction?
5µl for the template and 4µl for the primer.
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Why is DNA concentration and purity so important?
Because the quality of sequencing results depends greatly on the purity and concentration of the template DNA, it’s very important that you give careful attention to the preparation and quantitation of the DNA to be sequenced.
Highly concentrated plasmid preparations can have a detrimental effect on the electrophoresis of the sequencing reaction product. They can affect the machine capillaries and cause blockages, inhibiting the current and causing the reaction product to pass through a capillary slower than normal. This means that detection of these products starts later, resulting in retarded and poor quality results
Low DNA concentration leads to low raw data signals. When this happens, the software struggles to interpret the signal into a processed sequence. As a result, the sequence becomes unreliable.
The presence of multiple templates or primers in your samples will lead to reduced reliability and the presence of contaminants / impurities. They will also have the potential to inhibit everything from the sequencing reaction itself to the electrophoresis of the reaction products.
Common contaminants include salts (EDTA, NaCl, NaAc, Kac, KCl), proteins, detergents (SDS, Triton X-100), RNA, chromosomal DNA, organic chemicals (ethanol, chloroform, phenol), divalent cations (Mg, Ca, Mn), and excess PCR primers, dNTPs, enzyme and buffer components from PCR.
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Which stock sequencing primers can you provide?
‘Universal’ primers which are commonly found in cloning vectors are provided free of charge for use in automated sequencing. We have a list of all the sequencing primers the sequencing lab keeps in stock.
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How might I prepare samples?
Quantitation of template DNA
A Nanodrop device is available in D106. This provides a quick and accurate way of measuring template concentration and purity and only requires 1µl of sample.
Diluting the template to desired concentration
After determining the concentration, dilute the template to the correct final concentration using distilled water (please, no TE or other EDTA-containing buffer), and avoid adding any divalent cations (i.e. Mg, Ca, Mn).
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What chemistry and cycle sequencing program do you use?
We use Applied Biosystems’ BigDye (v3.1) Terminator chemistry. One primer and one template are mixed with DNA polymerase, a sequencing buffer, deoxynucleotides and dideoxynucleotides with specific fluorescent dyes attached to each base.
The DNA fragments with a dye labelled termination electrophorese pass through the polymer in the capillary, separating fragments by molecular weight. The DNA passes a detection window at the end of the capillary, where the fluorescent dyes are excited with a laser beam and emit light at wavelengths unique to each terminator.
A charged-coupled device (CCD) camera captures the emitted light, converts it to electronic information and transfers it to a computer. The computer software analyses the signal to generate sequence data and electropherograms.
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Can I ask for a special annealing temperature?
Standard reaction conditions are:
Denaturation (96°C), annealing (50°C) and extension (60°C) temperatures for 25 cycles.
A different program is used to sequence GC rich templates at denaturation (98°C), annealing (50°C) and extension (56.5°C) for 25 cycles.
Alternative sequencing reaction programs can be used if required.
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How do I view the sequencing files?
You can view the sequencing files (.ab1 files) using a free download (sequence scanner v2) from the Life tech website, unfortunately they make you register an account with them before you can download the software (see link below):
https://www.lifetechnologies.com/uk/en/home/life-science/sequencing/sanger-sequencing/sanger-dna-sequencing/sanger-sequencing-data-analysis.html
Alternatively you could use other programs, Chromas Lite and FinchTV (Google search), are quite popular in our department and are both free to download.
The .seq files will open in word.
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How do I interpret my sequence data?
We recommend that you always look closely at the electropherogram (.ab1 file) to make sure the sample provided has a strong enough signal to analyze correctly and enable accurate base calling. Accurate sequence assignment can only be made from the electropherogram; please don’t rely on the text file alone.
Electropherogram peaks should be sharp, well defined and evenly spaced. Signal intensities for nucleotides should be in the 200-800 range. Baselines should be uniform across the read. Sequence signal typically begins at a base of 20-40, however artifacts caused by unincorporated dye labelling compounds may be present as broad peaks (typically at base region 70-80 and also 100). These can cause the automated sequence-calling software to make incorrect assignments in these regions. Clean samples with relatively good signal strengths are usually not affected by such artifacts.
A sequence run from a sample at the appropriate level of purity and concentration, used with the correct primers, should generate sequences of read lengths at least 650-700 bases at accuracy of over 98%. Peak quality will diminish toward the end of a sequence run. We advise you not to use sequences to design subsequent primers from regions where the peaks become noticeably broad and less regularly spaced.
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Do you charge to rerun a reaction?
We don’t charge to rerun samples that might have failed due to potential machine or operator error.
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Do you offer a fragment analysis service?
Yes, the 3130xl Genetic Analyzer can be used for DNA fragment size analysis.
Assays include microsatellite, SNP and AFLP analysis. The instrument allows multiplexing study and the use of fifth dye technology. Size standards and dye sets we keep in house include:
- GS500 LIZ and GS500 ROX
- Dye Set G5: 6-FAM (Blue), VIC/HEX (Green), NED (Yellow), PET (Red), LIZ (Orange)
- Dye Set F: 5-FAM (Blue), JOE (Green), NED (Yellow), ROX (Red)
For more information regarding other dye sets and size standard available please refer to the Life tech website.
We do ask that users of the fragment analysis service contribute occasionally to the purchase of dye sets and size standards, particularly with large projects.
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