T1 Cloning Kit
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Price:
US$855.50
(Size: 20 rxns)
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T1 Cloning Kit is designed for cloning and sequencing Taq amplified PCR products. 5 minutes fast ligation of Taq-amplified PCR products. T7 promoter primer, M13 forward primer and M13 reverse primer for sequencing. T7 promoter for in vitro transcription.
Kit contents: | Component | 20 rxns | 60 rxns |
| T1 Cloning Vector (10 ng/µl) | 20 µl | 3 × 20 µl |
| Control Template (5 ng/µl) | 5 µl | 5 µl |
| Control Primer (10 µM) | 5 µl | 5 µl |
| M13 Forward Primer (10 µM) | 50 µl | 150 µl |
| M13 Reverse Primer (10 µM) | 50 µl | 150 µl |
| T1 Phage Resistant Chemically Competent Cells (abx098070) | 10 × 100 µl | 30 × 100 µl |
| Target |
T1 Cloning Kit |
| Storage |
Store Phage Resistant Chemically Competent Cells at -70°C for up to 6 months and all other components at -20°C for up to 9 months. |
| Availability |
Shipped within 10-20 working days. |
| Note |
This product is for research use only. This product is shipped with dry ice. |
| Directions for use |
Preparation: - Add 0.5 µl - 4 µl of PCR products and 1 µl of T1 Cloning Vector into a microcentrifuge tube.
- Gently mix, incubate at room temperature for 5 minutes, and place the tube on ice.
The optimal molar ratio of the vector to insert is 1:7 (for example, 1 kb = 20 ng, 2 kb = 40 ng). The optimal vector volume is 1 µl. The total reaction volume should be 3 µl - 5 µl. The optimal incubation temperature is 25 °C for most inserts, and may be higher (up to 37 °C) for others. The incubation time should be optimised by the end user, and the following recommendations may be used as a guide: - 0.1-1 kb: 5-10 mins
- 1-2 kb: 10-15 mins
- 2-3 kb: 15-20 mins
- ≥3 kb: 20-30 mins.
Note: If the insert is gel purified, use the maximum incubation time of 20-30 minutes. Transformation - Add the ligated products to 50 µl of T1 Phage Resistant Chemically Competent Cells and mix gently. Do not mix by pipetting up and down.
- Place on ice for 20-30 minutes.
- Heat shock the cells at 42°C for 30 seconds, then immediately place the tube on ice for 2 minutes.
- Add 250 µl of room temperature SOC or LB Medium, then incubate in a shaking incubator (200 rpm) at 37°C for 1 hour.
- Mix 8 µl of 500 mM IPTG and 40 µl of 20 mg/ml X-gal, and spread evenly on a selective LB plate. Incubate at 37 °C for 30 minutes.
- Inoculate a pre-warmed culture plate with 200 µl of the transformants.
Identification of positive clones - Transfer 5-10 white or light-blue colonies into 10 µl of Nuclease-free water and vortex.
- Use 1 µl of the mixture as the template for 25 µl PCR using M13 forward and M13 reverse primers.
- Carry out PCR using the following reaction conditions:
| Number of Cycles | Temperature | Time per Cycle | | 1 cycle | 94 °C | 10 min | | 30 cycles | 94 °C | 30 seconds | | 55 °C | 30 seconds | | 72 °C | Dependent on insert size and PCR Enzyme | | 1 cycle | 72 °C | 5-10 min | Note: PCR product size from vector self-ligation is 199 bp. Sequencing analysis Positive clones should be inoculated in Ampicillin or Kanamycin selective LB liquid media, then incubate in a shaking incubator (200 rpm) at 37°C for 6 hours. Isolate plasmid DNA for restriction enzyme digestion and DNA sequencing. Sequencing should be performed using M13 forward, M13 Reverse and T7 promoter. PCR of control insert (700 bp)Ligate 1 µl of control insert with 1 µl vector, and carry out PCR according to the following conditions: Required Components | Component | Volume | Final concentration | | Control template (5 ng/µl) | 1 µl | 0.1 ng/µl | | Control Primers (10 µM) | 1 µl | 0.2 µM | | 2X PCR SuperMix | 25 µl | 1X | | Nuclease-free water | Variable | - | | Total volume | 50 µl | - | PCR Conditions | Number of Cycles | Temperature | Time per Cycle | | 1 cycle | 94 °C | 2-5 min | | 30 cycles | 94 °C | 30 seconds | | 50-60 °C | 30 seconds | | 72 °C | 1 min | | 1 cycle | 72 °C | 10 min |
Notes - The PCR Enzyme should be a Taq DNA Polymerase.
- Primers must not be phosphorylated.
- 5-10 min post extension step is required. After amplification, agarose gel electrophoresis is recommended to validate the quality and quantity of the PCR products.
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