Sanger sequencing sample requirements
Sanger sequencing sample requirements
3730XL DNA Analyzer
Instrument by ThermoFisher (formally Applied Biosystem), a 96-capillary array DNA Sequencer for traditional Sanger Sequencing.
Specifications:
- Provides reads up to 1,000bp
- Low cost per sample
- Typical 24h turnaround time
Sample requirements
The DNA for this application needs to be very pure, purer than for manual sequencing. We recommend that the DNA should be prepared using a commercial kit such as Qiagen. Traditional methods such as alkaline lysis can work if proper care is taken.
Sample requirement per types:
- Plasmid DNA: concentration of 100ng/μl in a volume of 3μl per reaction* in water
- Cosmid DNA: concentration of 150ng/μl in a volume of 3μl per reaction* in water
- PCR fragments: concentration of 20ng per 100 base pairs (see table below) in a volume of 3μl per reaction* in water
(*) A minimum of 10μl volume is required to allow for evaporation or any other potential loss and any repeat reaction if necessary.
| PCR product | |
|---|---|
| Size | Concentration |
| 100-200 bp | 1-3 ng/ul |
| 200-500 bp | 3-10 ng/ul |
| 500-1,000 bp | 5-20 ng/ul |
| 1,000-2,000 bp | 20-40 ng/ul |
| >2,000 bp | 40-100 ng/ul |
Any non-standard primers submitted should be at a concentration of 10pm/μl (10μM) in water.
We use 3μl of primer solution per sequencing reaction, but please give us a minimum volume of 10μl to allow for evaporation or any other potential loss and any repeat reactions if necessary.
The correct amount of DNA as indicated above needs to be provided, we will not able to make any dilution due to amount of samples to process.
For user unsure about how to quantify their DNA, you can use a Nanodrop. This can give consistent, accurate results and automatically provides abs. 260/280 ratio (which for best results should be around 1.8).
DNA for sequencing should always be supplied in water only and not TE or Tris buffer. Also, please submit samples in tubes no smaller than 0.5ml to avoid handling problems.
Sample delivery
Samples and completed DNA sequencing request forms can be dropped off in the basket provided in the Biochemistry reception (Sanger Building), the department of Pathology reception or posted to the facility at the following address:
DNA Sequencing - CGS
Cambridge genomic services
Department of Pathology
Tennis court road
CB23 8EN Cambridge
Sequencing request form must be provided with a printed purchase order or the purchase order needs to be sent to cgs@path.cam.ac.uk
Stock primers
We can supply primers for use in sequencing reactions.
We also accept custom primers. Please design your sequencing primers to be between 18 and 22 bases long. Each primer should have a GC content between 50 and 60%. The primer annealing temperature should be between 55 and 60°C.
Please supply your custom primers at a concentration of 10pm/μl (10μM) in water. We use 2μl of primer solution per reaction but please give us an excess to allow for evaporation or any other potential loss.
| Primer | Sequence | Length |
|---|---|---|
| M13 forward (-20F) | 5'- TGT AAA ACG ACG GCCAGT - 3' | 18 |
| M13 reverse (-29R) | 5' - CAG GAA ACA GCT ATGAC - 3' | 17 |
| T7 | 5' - TAA TAC GAC TCA CTATAG GG - 3' | 20 |
| T7t | 5' - GCT AGT TAT TGC TCAGCG G - 3' | 19 |
| 5p6 | 5' - CGA TTT AGG TGA CACTAT AG - 3' | 20 |
| T3 | 5' - ATT AAC CCT CAC TAAAGG GA - 3' | 20 |
| pGEX 5' | 5' - GGG CTG GCA AGCCAC GTT TGG TG - 3' | 23 |
| pGEX 3' | 5' - CCG GGA GCT GCA TGTGTC AGA GG - 3' | 23 |
| -40F | 5' - GTT TTC CCA GTC ACGAC - 3' | 17 |
| -47F | 5' - CGC CAG GGT TTT CCCAGT CAC GAC - 3' | 24 |
| -48R (30nt_M13R) | 5' - AGC GGA TAA CAA TTTCAC ACA GGA - 3' | 24 |
| -55R | 5' - AAT TGT GAG CGG ATAACA ATT TCA - 3' | 24 |
| BGH reverse | 5' - TAG AAG GCA CAG TCGAGG - 3 | 18 |
| CMV forward | 5' - CGC AAA TGG GCG GTAGGC GTG - 3 | 21 |
Turnaround times
We realise that turnaround time is extremely important to our customers and aspire to provide
high quality results in the shortest possible time. Our current turnaround times are about 1 to 2
working days.