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 such as M13 Forward and Reverse, T3 and T7, which are commonly found in cloning vectors, are provided free of charge for use in automated sequencing. Suggestions for additional primers are welcome. Our currently held primers are:
T7P - TAA TAC GAC TCA CTA TAG GG
T7T - TAT GCT AGT TAT TGC TCA GCG G
M13F - GTA AAA CGA CGG CCA GT
M13R - GGA AAC AGC TAT GAC CAT G
M13-40 - GTT TTC CCA GTC ACG AC
Sp6 - ATT TAG GTG ACA CTA TAGT3
T3 - ATT AAC CCT CAC TAA AGG G
KS - TCG AGG TCG ACG GTA TC
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How might I prepare samples?
Possible protocols include:
Plasmids
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Qiagen Plasmid Mini/Midi/Maxi Kits or QIAprep Spin Miniprep Kit
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Promega Wizard Plus SV Miniprep (Do not use earlier versions of Wizard Plus Minipreps)
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Bio-Rad Quantum Prep or Aurum Plasmid Mini Kit
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Eppendorf Perfectprep Plasmid Mini
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GE Healthcare TempliPhi DNA Amplification Kit
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Modified Alkaline lysis/PEG precipitation method
PCR Products
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Millipore Montage PCR Centrifugal Filter Device or Microcon Centrifugal Filter Device
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Qiagen QIAquick PCR Purification Kit or QIAquick Gel Extraction Kit
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Shrimp Alkaline Phosphatase/Exonuclease I Treatment
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Ethanol Precipitation
Quantitation of template DNA
A Nanodrop device is available. 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?
Reactions are typically carried out in a thermal cycler, cycling between denaturation (96°C), annealing (50°C) and extension (60°C) temperatures for 25 cycles. Another program can be used to sequence GC rich templates at denaturation (98°C), annealing (50°C) and extension (56.5°C) for 25 cycles.
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How do I interpret my sequence data?
We recommend that you always look closely at the chromatogram to make sure the sample provided has a strong enough signal to analyse correctly and enable accurate base calling. Accurate sequence assignment can only be made from the chromatogram, please don’t rely on the text file alone.
Chromatogram 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 chromatogram. Sequence signal typically begins at a base of 20-40, however artefacts 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 artefacts.
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 650-700 bases at an 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 failed due to possible machine error. This is because as the capillary arrays age, they are more subject to individual errors.
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What are some of the common factors affecting the quality of sequencing results?
1. Inadequate template concentration
Templates that have too low a concentration will compromise base calling due to poor resolution of genuine base signals from background noise. Too high a concentration can interfere with electrophoretic migration, cause capillary blockage or saturation of the detection system with too high a signal. Details regarding template quantity can be found on the submission form.
2. Template purity
Samples that are mixtures of more than one template which share common priming sites will give mixed signals. However, samples containing a single species of DNA may still not yield adequate results. This is most commonly due to the inclusion of other reagents which may either interfere with the initial PCR reactions (for instance by inhibiting the Taq polymerase) or by compromising the subsequent electrophoresis. You can see a list of common contaminants here .
3. Primer quality
Please take care when designing custom primer oligonucleotides so that they don’t include mismatches and have a unique binding site on the template. Degeneracy or mismatches at or near the 3' end of the primer will probably result in a failed sequence run. In addition, they should not form secondary structures (particularly at the 3' end) and be designed to have a Tm adapted to a PCR annealing temperature of 50ºC.
Primers with a lower Tm will not hybridise to the complementary template efficiently, resulting in fewer extending fragments being generated. We recommend adding additional bases to the primer design if necessary to produce a sequence with a Tm in the region of 52 to 58ºC.
4. Difficult DNA content
Occasionally sequence runs fail even though all the above criteria are met. This may be due to the template DNA forming secondary structures (e.g. homopolymer hairpin loops which compromise the correct functioning of the Taq polymerase during PCR cycling) or DNA primary structure (e.g. AT or GC rich regions). In addition, DNA with reoccurring repeats or cosmid DNA may be difficult to sequence. No single chemistry will work on all possible sequencing experiments. Please see our guide to troubleshooting commonly encountered problems.
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Do you do fragment analysis?
Our 3130xl genetic analyser can also 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 (four reporter dyes plus a size standard). The size standard and dye sets available are:
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GS500 LIZ and GSROX 500
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Dye Set G5: 6-FAM (Blue), VIC/HEX (Green), NED (Yellow), PET (Red), LIZ (Orange, standard GS500 ROX)
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Dye Set F: 5-FAM (Blue), JOE (Green), NED (Yellow), ROX (Red, standard GS500 ROX)
To submit fluorescently labelled PCR fragments in tubes or 96 well plates, please use the submission form . Samples should be dried and properly labelled. Due to the nature of this type of work, it’s expected that you will have properly researched your particular project and will request the appropriate standards set. Results will normally be emailed to you within three working days.
The peak scanner software can be downloaded and enables you to view, edit, analyse, print and export fragment analysis data from the genetic analyser. Samples which fail to give data due to obvious mechanical failure will be rerun free of charge.
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