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| Product Name | M13 Forward and Reverse Universal Primer Set for Sequencing |
| Catalog No. | PRIPRO-0021 |
| Description | A paired set of M13 forward (M13-F, 5′-GTAAAACGACGGCCAGT-3′) and M13 reverse (M13-R, 5′-CAGGAAACAGCTATGAC-3′) universal sequencing primers. These 17-mer oligonucleotides are the most widely used universal primers for Sanger sequencing of plasmid clones containing M13-derived priming sites (commonly found in pUC, pBluescript, pGEM, and related vectors). The primers flank the multiple cloning site (MCS) of standard cloning vectors, enabling bidirectional sequencing of inserted DNA fragments. Both primers are desalted and supplied at a ready-to-use concentration for direct addition to sequencing reactions. Each primer set is quality controlled by mass spectrometry and functional sequencing verification. |
| Intended Use | Designed for Sanger sequencing of plasmid DNA containing M13 priming sites; also suitable for colony PCR screening of recombinant clones, site-directed mutagenesis verification, and as universal forward/reverse primers for PCR amplification of inserts from MCS-flanked vectors. |
| Principle / Technology | Sequence-specific priming at regions immediately flanking the multiple cloning site in common plasmid vectors; M13-F primes from the lacZ/lac promoter side, M13-R primes from the opposite strand |
| Detection Method | Sanger dideoxy sequencing with fluorescent terminators; capillary electrophoresis on ABI or equivalent sequencers; PCR amplification for clone screening by agarose gel electrophoresis |
| Sample Type | Purified plasmid DNA from bacterial clones containing vectors with M13 priming sites (pUC18/19, pBluescript II SK/KS, pGEM-T, pCR2.1, etc.) |
| Performance Range / Specifications | 17-mer primers; Tm ~52–55 °C; GC content ~47%; sequencing read length typically 500–800 bases of high-quality read from standard plasmid templates |
| Sensitivity / LOD | Reliable sequencing from 200–500 ng plasmid DNA per reaction; colony PCR detection from single bacterial colony template |
| Specificity | Highly specific to M13 priming sites; minimal cross-priming with bacterial genomic DNA; sequencing background typically <5% of total signal |
| Reaction Conditions / Protocol | For sequencing: 3.2 pmol primer per 10 µL reaction with 200–500 ng plasmid template and BigDye Terminator chemistry. For colony PCR: 0.2–0.5 µM each primer with Taq polymerase and single colony template. |
| Components / Formulation | Two tubes: M13 Forward (17-mer) and M13 Reverse (17-mer); each supplied as lyophilized or pre-dissolved at 5 µM (for sequencing) or 100 µM stock |
| Storage Conditions | –20 °C; stable in solution; protect from repeated freeze-thaw |
| Shelf Life | 24 months at –20 °C; 12 months at 2–8 °C |
| Package Specifications | Two tubes containing 1–5 nmol each of M13-F and M13-R; supplied as a matched set |
| Product Form | Lyophilized white pellet or clear solution |
| Quality Control | MALDI-TOF MS mass confirmation for each primer; HPLC or PAGE purity ≥90%; functional QC by Sanger sequencing of control plasmid (>500 bp read length, >98% base calling accuracy); DNase/RNase-free. |
| Key Features | Universal primers for most cloning vectors; bidirectional sequencing coverage of inserts; economical ready-to-use format; applicable for both sequencing and colony PCR; MS-verified sequence integrity; matched F/R set in one order. |
| Purity | ≥90% by HPLC; mass confirmed by MALDI-TOF MS; single 17-mer species |
| Concentration | Pre-dissolved at 5 µM (sequencing-ready) or supplied as 100 µM stock; lyophilized option for custom resuspension |
| Activity / Unit Definition | Efficiently primes Sanger sequencing with >500 bp read length; colony PCR amplification of inserts up to 5 kb; functional in standard sequencing and PCR buffer systems |
| Molecular Weight | M13-F: ~5,200 Da (17-mer); M13-R: ~5,150 Da (17-mer); exact mass per batch reported on COA |
| Source / Origin | Synthetic phosphoramidite chemistry; sequence based on M13mp18 bacteriophage genome |
| pH Range / Optimal pH | Stable and functional pH 6.0–9.0; optimal in sequencing buffer pH 8.5–9.0 |
| Shipping Conditions | Ambient temperature; lyophilized primers highly stable during transit |
| Expiration Date / Stability | 24 months at –20 °C dry or in TE; 12 months at 2–8 °C; working dilutions stable for 6 months at –20 °C |
| Regulatory / Compliance | For research use only; not for diagnostic applications |
| Compatibility | Compatible with BigDye Terminator v1.1 and v3.1 sequencing chemistry; all ABI capillary sequencers (310, 3100, 3130, 3730 series); all standard Taq polymerases for colony PCR |
| Recommended Buffer System | Nuclease-free water or TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0); sequencing dilution buffer available upon request |
| Application Notes / Precautions | For sequencing, verify primer-to-template ratio: too little primer reduces signal, too much increases background. For GC-rich templates, consider adding 5% DMSO or using betaine. Colony PCR: touch a sterile tip to colony, swirl in PCR mix, then streak a reference plate. |
| Batch-to-Batch Consistency | Sequencing read length within ±50 bases across batches; base calling accuracy >98%; MS mass within ±0.05% theoretical |
For research use only, not for clinical use.
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