1. pSWE-Topo Zero Cloning Kit, 25 T, for screening positive recombinants by the expression of suicide gene. $500

Description

Product Information

Description

The pSWE-Topo Zero Cloning Kit produced by our company is a vector for screening positive recombinants by the expression of suicide gene. It is transformed from 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 correctly expressed, and the cells containing the vector can grow normally. This vector is also known as the “Zero” background vector. At the same time, the pSWE-Topo Zero Cloning Kit uses a new vector ligation method, which does not require additional ligase for the ligation reaction between the fragment and the vector. It only needs to add the target gene fragment to the vector to be reacted for 5 min to carry out the conversion operation at room temperature (22 ℃-37 ℃). The 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 enzymatic ligation is required, the target gene fragment is added to the system, and normal transformation can be performed for 5 minutes at room temperature; the positive rate can reach 95%; short and long fragments All clones are suitable; the selection marker is ampicillin;

sequencing primers: M13 Forward Primer, M13 Reverse Primer.

Storage and Handling Conditions

Transported with wet ice; stored at -20°C, valid for 12 months.

Component

Assay Protocol

1. Preparation of PCR Products

(1) The primers cannot be phosphorylated; (2) High fidelity and Taq DNA Polymerase can be used for the PCR reaction; (3) It is recommended to use a gel recovery kit for purification of PCR products after electrophoresis.

2. Reference reaction system:

The recommended amount 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 a 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. Conversion:

(1) Add 50-100 μL of clone competent (or add the reaction product to 50-100 μL of clone competent) into the reaction system, mix gently, and place in ice for 30 minutes;

(2) The transformation system was immediately heat-shocked at 42°C for 45 s, and immediately placed on ice for 2-3 min;

(3) Add 200 μL of SOC or LB medium equilibrated to room temperature into the transformation system, and incubate at 200 rpm and 37 °C for 1 h;

(4) Take 200 μL of bacterial liquid to coat the resistant plate, and invert at 37°C overnight (in order to obtain more clones, part of the supernatant can be removed after centrifugation, and all the bacterial liquid is coated on the resistant plate).

5. Detection of positive transformants:

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

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

(3) Sequencing to identify positive clones: use M13 Forward Primer and M13 Reverse Primer primers to sequence and analyze.

Note:

1. It should be stored in an ice box with using this product, and it should be stored at -20°C immediately after use.

2. Escherichia coli strains DB3.1, ccdB Survival, Stable, JM109, XL1-Blue and XL10-Gold are resistant to CcdB, and they can be used for reproduction or preservation of ccdB-containing plasmids, so they are not suitable for pSWE-Topo Zero Vector. filter. Competent cells of Escherichia coli that are intolerant to Ccdb, such as DH5α, Top10, TG1, which are commonly used in other laboratories, are suitable.

3. For your safety and health, please wear a lab coat and disposable gloves.

For Research Use Only!

Ver. No.: V1.0-202111