Principle of pBLUE-T Simple Vector Rapid Cloning Kit T4

pBLUE-T SimpleVector rapid cloning kit

Principle of pBLUE-T Simple Vector Rapid Cloning Kit T4

Catalog Number:42119

Composition, storage, and stability of the reagent kit:

Storage at -20 ℃ will not affect the effectiveness of use within 6 months.

Product Introduction

PBLUE-T Simple Vector is a specialized vector for efficient cloning of PCR products (TA Cloning). This vector is modified from the pBlueScript II SK (+) plasmid. Unlike the traditional T vector ECOR V with T addition after cleavage, pSURE-T Simple modifies the pBlueScript II SK (+) by introducing Xcm I enzyme cleavage at the original pBlueScript II SK (+) site, resulting in unpaired T bases directly at the 3 'ends on both sides of the original site, thus achieving higher recombination efficiency. At the same time, it eliminates the multi clone enzyme cleavage sites on the pBlueScript II SK (+) vector, requiring the introduction of suitable enzyme cleavage sites on PCR amplification primers. At this point, if the enzyme cleavage site introduced by PCR amplification primers is used for DNA cleavage, the cleavage reaction will not be affected by restriction enzymes on other polyclonal cleavage sites on the T vector, which can greatly improve the efficiency of cleavage and increase the success rate of subcloning. In addition, our carrier connection system has the characteristics of low background (less than 10% blue spots), high recombination rate (over 90% of white spots have inserted fragments), and fast connection. The technical information related to the pBLUE-T Simple vector is listed at the end of this manual. The complete sequence can refer to the pBlueScript II SK (+) sequence, but the sequence at the multi clone enzyme cleavage site is slightly different.

It is recommended to use M13 universal sequencing primers and T3 promoter primers for sequencing (see diagram below)
Operation steps:
1. Preparation for Connection Reaction

Whether PCR products need to be purified depends on the quality of the amplified products. If the PCR product is very clean, the ligation reaction can be carried out directly without purification. But if the PCR product is based on a plasmid template, it must be purified, and the template plasmid may also form white spots. PCR products can be separated by agarose gel electrophoresis. The DNA product rapid purification and recovery kit produced by our company can effectively recover DNA fragments of over 70bp.

The PCR products amplified by Taq, Tth, AmpliTaq, and KlenTaq DNA polymerases all have a prominent 3 'at the end-A. Having 3 '-The PCR product at the A-terminus can be directly cloned and ligated using the pBLUE-T Simple vector. Having 3 '®The PCR product amplified by DNA polymerase with 5 'exonuclease activity (high fidelity enzyme) is a flat end. To clone this flat end PCR product, a 3' - step should be taken first-End to end work with A.

2. Conventional Connection Reaction:

1) On a standard 10mConnect the reaction system and add 1mL 30% of pblue-t simplemPCR products (under normal circumstances, it is not necessary to accurately quantify PCR products. Generally, optimizing the molar ratio of PCR products to carriers to 2:1~10:1 can achieve good results, with a recommended ratio of 3:1), 1ml 10´Alignment Buffer and 0.5-1mT4 DNA Ligase of l (2.5-5 Weiss Units), supplemented with water for the rest. The reaction proceeds according to the following system:

Usually, T4 DNA Ligase is added at the end.

2) Connect overnight at 16 ℃ (usually done on a PCR instrument).

It is usually recommended to connect overnight at 16 ℃ (10 ℃)mThe standard amount of ligase in the system is 2.5 Weiss Units, which can obtain the most transformants. However, the inclusion of PEG Enhancer in this system can improve the efficiency of ligation several times, especially at high levels of ligase (10)mThe recommended amount of ligase for the L system is 5 Weiss Units. Connecting at 16 ℃ for 30 minutes can meet the requirements of general research.

3) Cool on ice and then convert or store at -20 ℃.

3. Quick connection response:

1) The reaction proceeds according to the following system:

Usually, T4 DNA Ligase is added at the end.

2) Connect at 22 ℃ for 5-10 minutes (usually done on a PCR instrument).

2 ´The Quick Ligation Buffer already includes all optimized quick connect components, and it is usually recommended to connect at 22 ℃ for 10 minutes (10 minutes)mThe amount of ligase in the L system is 5 Weiss Units, and long fragment ligation can be extended to 30 minutes, which generally yields satisfactory results. In addition, this system can also be connected for 30 minutes at 16 ℃ (10mThe amount of ligase in the L system is 5 Weiss Units, which can meet the requirements of general research.

3) Cool on ice and then convert or store at -20 ℃.

4. Conversion:

at 50-100mL competent cells were placed on ice, and after thawing, they were lightly brushed several times to evenly suspend the cells.

at Join 4-5mConnect the liquid (up to a maximum of all can be added, as long as the volume does not exceed 1/10 of the volume of the competent cells), gently mix well. Leave it on ice for 30 minutes. Heat shock in a 42 ℃ water bath for 90 seconds. Leave on ice for 2-3 minutes.

at Add 500mLB or SOC medium (without antibiotics), shake at 37 ℃ and 150 rpm for 60 minutes.

at Take 200mBacteria coated in advance with 16m50mg/ml IPTG and 40mOn a ampicillin su plate coated with 20 mg/ml X-gal.

The amount of bacteria to be coated should be adjusted appropriately based on the efficiency of the connection and the sensitivity of the competent cells. If the expected number of clones is small, a portion of the culture medium can be removed by centrifugation (4000rpm, 2 minutes), leaving an appropriate amount of culture medium to suspend the bacterial cells. Then, all or an appropriate amount of the bacterial cells can be coated on a plate (the remaining bacterial solution can be stored at 4 degrees Celsius, and if the number of transformed colonies is small on the second day, all the remaining bacterial solution can be coated on a new culture plate).

at Place the plate forward at 37 ℃ for 1 hour to absorb excess liquid, then invert and culture overnight.

5 Filter:

1). Blue white screening of transformants:

When an exogenous DNA fragment is inserted into pBLUE-T Simple, the nucleic acid sequence of the exogenous DNA alters the coding of the LacZ gene, thereby affecting its productb-galactosidasea-The activity of the fragment resulted in the recombinant clone appearing white on the X-gal/IPTG plate, while the non recombinant clone appeared blue. Sometimes the insertion of fragments does not affect the reading frame of the lacZ gene, or the insertion fragment is too small. In this case, the colony (recombinant clone) appears light blue or a light blue spot in the center of the colony, with a white outer ring (fish eye shaped blue spot). Select white or light blue colonies grown on IPTG/X-gal plates, pick them with a toothpick onto liquid medium containing ampicillin su, and incubate overnight at 37 ℃.

2). Identification of transformants:

a. Extract the plasmid with the liquid of the white colony cultured above, digest it with the restriction site of the inserted segment, and check the size of the segment by agarose gel electrophoresis to determine whether it contains the target segment.

b. Select white bacterial colonies for direct PCR detection

c. Use T3 promoter primers or M13 universal sequencing primers for sequencing to determine if the target clone is present.

at How to calculate the amount of PCR product required for the linkage reaction?

Generally, optimizing the molar ratio of PCR product to vector to 2:1~10:1 (recommended 3:1) can yield good results. The following formula can be used:

[Amount of vector added (ng) x size of inserted fragment (kb) ÷ size of vector (kb)] x molar ratio of inserted fragment to vector=amount of inserted fragment (ng). For example, the molar ratio of inserted fragment to vector is 3:1. If 40ng of vector is added in the ligation reaction and the size of inserted fragment is 1000bp, the amount of inserted fragment should be [40ng vector x 1kb inserted fragment ÷ 2.928kb vector] x 3/1=40.1ng.

Principle of pBLUE-T Simple Vector Rapid Cloning Kit T4