T1 Cloning Kit

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Catalogue No: abx098055
Price: US$855.50
(Size: 20 rxns)
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Plasmid map (T1).

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Datasheet SDS
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:
  1. Add 0.5 µl - 4 µl of PCR products and 1 µl of T1 Cloning Vector into a microcentrifuge tube.
  2. 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

  1. 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.
  2. Place on ice for 20-30 minutes.
  3. Heat shock the cells at 42°C for 30 seconds, then immediately place the tube on ice for 2 minutes.
  4. Add 250 µl of room temperature SOC or LB Medium, then incubate in a shaking incubator (200 rpm) at 37°C for 1 hour.
  5. 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.
  6. Inoculate a pre-warmed culture plate with 200 µl of the transformants.

Identification of positive clones

  1. Transfer 5-10 white or light-blue colonies into 10 µl of Nuclease-free water and vortex.
  2. Use 1 µl of the mixture as the template for 25 µl PCR using M13 forward and M13 reverse primers.
  3. 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.
Research Articles on T1 Cloning Kit


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