Sanger sequencing
As the Gold standard of DNA sequencing, Sanger sequencing remains ideal for small-scale projects and for validation. We offer sequencing starting from various DNA templates or directly from the sequencing reaction.
Intro
With longer reads and an extremely low error rate, Sanger sequencing remains the best option for low-throughput sequencing and for validation of NGS results. Sequencing of DNA fragments based on the Sanger method relies on the selective incorporation of labeled dideoxynucleotide chain-terminators during in vitro DNA replication followed by automated capillary electrophoresis. The four bases are detected using different fluorescent labels. These are detected and represented as “peaks” of different colors that can then be interpreted to determine the base sequence. At the GIGA Genomics platform, we use the ABI3700 automated capillary gel electrophoresis system from Applied Biosystems.
Applications
We proposed two different services: run only or sequencing reaction+run.
Possible DNA templates are plasmids, PCR amplicons, phages and bacterial artificial chromosomes (BAC).
In practice
Run only
Samples
10μl of sequencing reaction product not purified/sequence reaction
• sent to the genomic platform (GIGA, B34, 1st floor), in tubes, strips or 96 plates.
• clearly identify
• covered with foil.
If possible, a positive control will be used for the sequencing reaction.
Delay
2-3 working days depending on the number of samples to analyse
Deliverable
Chromatogram and text sequence in a *.abi file and a QC report from Sequence Scanner with our controls will be put on a server with a personal access.
The results will be available on this server for 3 months.
Form
Sequencing reaction+run
Samples
Purified and diluted DNA (for purification service before sequencing, see below)
• resuspended in water or Tris 10mM pH8.0 (NO EDTA)
• sent to the genomic platform (GIGA, B34, 1st floor), in 0,2 to 1,5 ml tubes
• clearly identify
Minimum volume of 10μl/sequence reaction
EDTA has to be avoided!
* For BAC sequencing, the DNA must be provided at a concentration around 50 to 100 ng/µL and a volume around 15 to 20µL per sequence. The primer must absolutely be provided seperately at a concentration of 10µM (quantity: 10µL/sequence). The Tm of the primer must be >55°C.
Quantification: if you quantify your phage DNA using the nanodrop, don't forget that the constant is 37 and not 50!
Primers should be provided by the customer:
• Resuspended in water
- Concentration : 5 to 10 μM
- Volume : 10 μl/sequence reaction
• Tm between 50°C and 60°C
• No secondary structure
• With a unique site for the annealing
The GIGA Genomics platform also provides universal primers:
M13 FW CGC CAG GGT TTT CCC AGT CAC GAC
M13 RV TCA CAC AGG AAA CAG CTA TGA C
BGHreverse TAG AAG GCA CAG TCG AGG
SP6LONG ATT TAG GTG ACA CTA TAG AAT AC
SP6 promoteur TAT TTA GGT GAC ACT ATA G
T3 promoteur ATT AAC CCT CAC TAA AGG GA
T7LONG GTA ATA CGA CTC ACT ATA GGG C
T7 promoteur TAA TAC GAC TCA CTA TAG GG
T7term CTA GTT ATT GCT CAG CGG T
CMVfor CGC AAA TGG GCG GTA GGC GTG
PJET 1.2 FW CGA CTC ACT ATA GGG AGA GCG GC
PJET 1.2 RW AAG AAC ATC GAT TTT CCA TGG CAG
Delay
3-4 working days depending on the number of samples to analyse (4-5 working days if purification is required as additional service)
Deliverable
Chromatogram and text sequence in a *.abi file and a QC report from Sequence Scanner with our controls will be put on a server with a personal access.
The results will be available on this server for 3 months.
Form
