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pSWE-Topo Zero Cloning Kit for DNA connection molecular biology reagent

specification:25T
  • G3022-25T
  • Servicebio
  • Servicebio

Product Description

G3022-25T

Introduction:

The pSWE-Topo Zero Cloning Kit produced by our company is a vector for screening positive recombinants through the expression of suicide genes and is modified from the pUC19 vector. When the target gene fragment is not connected to the vector, the suicide gene is successfully and correctly expressed, and the cells containing the vector cannot grow; when the target gene fragment is successfully connected to the vector, the suicide gene will not be expressed correctly, and the cells containing the vector can grow normally. This vector is also called "Zero" background vector. At the same time, our company's pSWE-Topo Zero Cloning Kit uses a new vector ligation method, no additional ligase is needed to connect the fragment to the vector, only the target gene fragment needs to be added to the vector at room temperature (22℃-37 ℃) The conversion operation can be carried out after reacting for 5 minutes. pSWE-Topo Zero Cloning Kit is compatible with TA cloning and blunt-end cloning.

 

Features: Easy to use, compatible with TA cloning and blunt-end cloning; no additional enzyme reaction is required, the target gene fragment is added to the system, and normal transformation can be carried out within 5 minutes at room temperature; the positive rate can reach 95%; short and long fragments Applicable for cloning; selection marker is ampicillin; sequencing primers: M13 Forward Primer, M13 Reverse Primer.

 

Storage and transportation

Transport with wet ice; storage at -20°C, valid for 12 months.

Component Number                

Component                

G3022-25T                

G3022-1

pSWE-Topo Zero Vector (50 ng/μL)

25 μL

G3022-2

Control Template (700 bp, 50 ng/μL)

10 μL

G3022-3

M13 Forward Primer (10 μM)

50 μL

G3022-4

M13 Reverse Primer (10 μM)

50 μL

Manual

1

 

Steps

1. Preparation of PCR products

(1) Primers cannot be phosphorylated; (2) High-fidelity and Taq DNA Polymerase can be used for PCR reactions; (3) It is recommended to use gel recovery kits for purification of PCR products after electrophoresis.

2. Reference reaction system:

Component                

Volume                

pSWE-Topo Zero Vector (50 ng/μL)

1 μL

Gene fragment

0.5-4 μL

Recommended dosage of insert: the molar ratio of vector to fragment = 1:10-1:3. It can be roughly calculated by adding 50 ng of 1 kb fragment. If the gene library is constructed, the reaction system can be appropriately expanded.

3. Gently mix the vector and gene fragments according to the above reference reaction system, react at room temperature (20-37°C) for 5-10 minutes, and place the centrifuge tube on ice.

4. Transformation: a. Add 50-100 μL clone competent (or add the reaction product to 50-100 μL clone competent), mix gently, and ice bath for 30 min; b, immediately transform the system Heat shock at 42°C for 45 s, immediately place on ice for 2-3 min; c, add 200 μL of SOC or LB medium equilibrated to room temperature in the transformation system, and incubate at 200 rpm and 37°C for 1 h; d, take 200 μL Spread the bacterial solution on a resistant plate and invert it overnight at 37°C (in order to obtain more clones, you can remove part of the supernatant after centrifugation and spread the bacterial solution on the resistant plate).

5. Detection of positive transformants:

a. Colony PCR identification of positive clones: pick a single clone into 10 μL of sterile water, vortex to mix, then take 1 μL of the mixture as a PCR template, and M13 Forward Primer and M13 Reverse Primer as universal primers to identify positive clones by colony PCR . (Recommend G3304 and G3305; refer to the corresponding product manual for PCR reaction system and procedures, and PCR amplified fragments of positive clones> 100 bp)

b. Identification of positive clones by restriction enzyme digestion: pick a single clone and inoculate it in an appropriate amount of resistant liquid medium, and cultivate it overnight at 220 rpm and 37°C. A small amount of plasmid was extracted, digested with appropriate restriction enzymes, and positive clones were identified by gel electrophoresis.

c. Sequencing to identify positive clones: use M13 Forward Primer and M13 Reverse Primer primers for sequencing and analysis.

 

Precautions

1. When using the product, it is advisable to store the product in an ice box or on an ice bath, and store it at -20°C immediately after use.

2. E. coli strains DB3.1, ccdB Survival, Stable, JM109, XL1-Blue and XL10-Gold, these strains can tolerate CcdB and can be used to propagate or store ccdB-containing plasmids, so pSWE-Topo Zero Vector is not applicable To filter. Competent cells of Escherichia coli that are not tolerant to Ccdb, such as DH5α, Top10, TG1, which are commonly used in other laboratories, are applicable.

3. For your safety and health, please wear lab coats and disposable gloves for operation.

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