Sanger Sequencing
With Sanger Sequencing, gel or column-purified PCR products and plasmids can be used as starting material. We also perform sequencing services for “Ancient DNA Samples”. Sequencing results can typically be obtained for upto 550 nucleotides for single direction sequencing, and upto 1100 nucleotides for dual direction sequencing. The resulting output of sequencing depends on the purity and quality of the starting material. If desired, we can design and synthesize suitable primers. With paired-end sequencing, it is possible to obtain more accurate results. In our lab, the ABI Prism 3130 Genetic Analyzer is used for sequencing services.
General Information
The working principle of the machine is the generation of a monochromatic light via a laser located within the electrophoretic units. The gel-matrix that the DNA is loaded into is scanned by the monochromatic light. In the electrophoresis step, when the region with the fluorescent dye-bound DNA is arrived at, a signal is given and a characteristic wavelength of light is scattered. This signal is received by the detector and the results obtained are assessed and then turned into sequence data.
Price
| PRICE (€) (UP TO 10 SAMPLES) | 10-199 SAMPLES | 200 OR MORE SAMPLES | |
|---|---|---|---|
| Sanger Sequencing of Plasmids | € 11.99 | € 10.99 | € 9.99 |
| Sanger Sequencing of PCR Products | € 11.99 | € 10.99 | € 9.99 |
| NGS Sequencing of Plasmids (max: 5 kb) | € 24.99 | ||
| NGS Sequencing of PCR products (max: 5 kb) | € 24.99 | ||
| PCR Product Purification | € 1 |
- NGS services are provided using PGM Ion Torrent™ ve Illumina MiSeq™.
- Other NGS services (RNASeq, WGS, etc.) are undertaken as independent projects. Please contact us for details.
Sample Preparation
- DNA samples and primers (sequencing order form should be filled out and sent to us).
- Concentration of primers to be used in sequencing (at least 10 μM concentration and at least 5 μL of primer/reaction) and sequence information (preferred).
- Concentration of DNA sample (for plasmids, at least 80 ng/μL and 5-10 μL/reaction is recommended; for PCR products, at least 10 ng/μL and 5-10 μL/reaction is recommended)
- Gel image and spectrophotometric measurement results (concentration, A260/280 and A260/230 values) for PCR products (run in 2% agarose gel) or plasmid, phage, cosmid or BAC DNA (run in 1% agarose gel).
Requirements for DNA Sequence sample delivery
- Sentebiolab accepts both purified and unpurified PCR products and plasmid DNA
- To ensure optimal concentration, we recommend the values shown in the table below
- To ensure that there is only one band in purified PCR products, they should be checked by gel electrophoresis.
- Samples must be clearly labeled.
| SAMPLE | CONCENTRATION | VOLUME / RXN |
|---|---|---|
| Plasmid | 200 ng/μl | 10 μl |
| PCR products | 50 ng/μl | 10 μl |
| Primer | 10 μM | 10 μl |
- All of your samples, including the primers, should be provided in 1.5 mL, 0.5 mL or 0.2 mL tubes and should be labeled in the manner in which you wish the sample to be identified.
- Sentebiolab is not responsible for problems such as leakage, evaporation or cross-well contamination (due to the samples not being prepared in the proper manner) or problems arising during delivery.
Frequently Used Primers
| PRIMER NAME | SEQUENCE |
|---|---|
| 5’AD | 5′- AGG GAT GTT TAA TAC CAC TAC -3′ |
| 3’AD | 5′- AGA TGG TGC ACG ATG CAC AG -3′ |
| 5AOX1 | 5′- GAC TGG TTC CAA TTG ACA AGC -3′ |
| 3AOX1 | 5′- GCA AAT GGC ATT CTG ACA TCC -3′ |
| A-FACTOR | 5′-TAC TAT TGC CAG CAT TGC TGC -3′ |
| 3’BD | 5′- TAA GAG TCA CTT TAA AAT TTG TAT C -3′ |
| CMV-Forward | 5′- CGC AAA TGG GCG GTA GGC GTG -3′ |
| CMV-R | 5′- GTT CAC GGT GCC CTC C -3′ |
| PGEX-5′ | 5′- GGG CTG GCA AGC CAC GTT TGG TG -3′ |
| PGEX-3′ | 5′- CCG GGA GCT GCA TGT GTC AGA GG -3′ |
| T3 | 5′- ATT AAC CCT CAC TAA AGG GA -3′ |
| T7 | 5′- TAA TAC GAC TCA CTA TAG GG -3′ |
| T7-Ter | 5′- GCT AGT TAT TGC TCA GCG G -3′ |
| BGH | 5′- TAG AAG GCA CAG TCG AGG -3′ |
| SP6 | 5′- GAT TTA GGT GAC ACT ATA G -3′ |
| M13F | 5′- TGT AAA ACG ACG GCC AGT -3′ |
| M13R | 5′- CAG GAA ACA GCT ATG AC -3′ |
| M13F(-47) | 5′- CGC CAG GGT TTT CCC AGT CAC GAC -3′ |
| M13R(-48) | 5′- AGC GGA TAA CAA TTT CAC ACA GGA -3′ |
| GLP1 | 5′- TGT ATC TTA TGG TAC TGT AAC TG -3′ |
| GLP2 | 5′- CTT TAT GTT TTT GGC GTC TTC CA -3′ |
| pBAD Forward | 5′- ATG CCA TAG CAT TTT TAT CC -3′ |
| pBAD Reverse | 5′- GAT TTA ATC TGT ATC AGG -3′ |
| pEGFP-N-5′ | 5′- TGG GAG GTC TAT ATA AGC AGA G -3′ |
| pEGFP-N-3′ | 5′- CGT CGC CGT CCA GCT CGA CCA G -3′ |
| pEGFP-C-5′ | 5′- CAT GGT CCT GCT GGA GTT CGT G -3′ |
| pEGFP-C-3′ | 5′- TAT GGC TGA TTA TGA TCA GT -3′ |
| pFastBacF | 5′- GGA TTA TTC ATA CCG TCC CA -3′ |
| pFastBacR | 5′- CAA ATG TGG TAT GGC TGA TT -3′ |
| RVP3 | 5′- CTA GCA AAA TAG GCT GTC CC -3′ |
| RVP4 | 5′- GAC GAT AGT CAT GCC CCG CG -3′ |
| PMAL-C2X-F | 5′- TGC GTA CTG CGG TGA TCA AC -3′ |
| PMAL-C2X-R | 5′- CTG CAA GGC GAT TAA GTT GG -3′ |
| PQE30F | 5′- TGA GCG GAT AAC AAT TTC AC-3′ |
| PQE30R | 5′- GTT CTG AGG TCA TTA CTG G-3′ |
| S.TAG | 5′-CGA ACG CCA GCA CAT GGA CA-3′ |