PBLUE-T Vector Cloning Kit T4 Principle Vector and Others

NoboledePBLUE-T Vector Cloning Kit T4 Principle Vector and Others

PBLUE-T Vector Cloning Kit

Catalog Number: 42014

Composition, storage, and stability of the reagent kit:

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

The specific product parameters are subject to the parameters in the product manual received

Product Introduction

Many high-temperature DNA polymerases, such as Taq DNA polymerase and Tth DNA polymerase, amplify PCR products at 3 '-The PCR product with a prominent base A at the end can be used with 3 '-A vector with a prominent base T at the end facilitates cloning. The pBLUE-T vector is derived from the pBlueScript II SK (+) plasmid, with an appropriate sequence added at the EcoRI cleavage site. After XCM I cleavage, an unpaired T base is directly generated at its 3 'end, resulting in higher recombination efficiency. The two ECOR I sites independently designed at both ends of the pBLUE-T vector insertion site enable the insertion fragment to be detected by inexpensive and efficient ECOR I single enzyme digestion; Meanwhile, the pBLUE-T vector does not contain NdeI or NcoI restriction enzyme sites, making it convenient for cloning genes containing the aforementioned enzyme sites. 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 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.

Sequencing can be performed using T3, T7 promoter primers and M13 universal sequencing primers (see map 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. PCRThe products can be separated by agarose gel electrophoresis. The DNA product rapid purification and recovery kit produced by our company is suitable for 7DNA fragments larger than 0bp can be well recovered.

The PCR products amplified by Taq, Tth, AmpliTaq, and KlenTaq DNA polymerases all have a prominent 3 'at the end-A. have3’-The PCR product at the A-terminus can be directly cloned and ligated using the pBLUE-T vector. Having 3 '®The PCR product amplified by DNA polymerase with 5 'exonuclease activity (high fidelity enzyme) is a flat end PCR product. To clone this flat end PCR product, it should be performed with a fine 3' line-End to end work with A.

2. Conventional Connection Reaction:

1) On a standard 10mConnect the reaction system and add 1ml30% of pblue-tCarrier Xml PCRProduct (under normal circumstances, there is no need forPCRAccurate quantification of the product can be achieved by optimizing the molar ratio of PCR product to carrier to 2:1~10:1, with a recommended ratio of 3:11ml 10´ligation BufferAnd 0.5-1ml(T4 DNA of 2.5-5 Weiss UnitsLigase， Make up the remaining water. 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 ℃)mlThe standard amount of ligase in the system is 2.5Weiss UnitsIt can obtain the maximum number of transformants. But the PEG Enhancer in this system can improve the connection efficiency several times，Under high levels of ligase (10)mlThe recommended amount of ligase for the system is 5Weiss 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（10mlThe amount of ligase in the system is 5Weiss UnitsLong segment connections can be extended up to 30 minutes and generally yield satisfactory results. In addition, this system can also be connected for 30 minutes at 16 ℃ (10mlThe amount of ligase in the system is 5Weiss Units can meet the requirements of general research.

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

4. Conversion:

ð50-100mL competent cells are placed on ice, and after thawing, gently brush several times to evenly suspend the cells.

ðJoin 4-5mlConnect the solution (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. Placed on ice30 Minutes. 42 ℃ water bath heat shock 90In seconds. Place 2 on ice~3 minutes.

ðAdd 500mLB or SOC medium (without antibiotics), 37 ℃ 150 rpmShake and cultivate for 60 minutes.

ðTake 200mlBacteria coated in advance with 16ml 50mg/ml IPTGAnd 40mOn a ampicillin agar 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).

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

1). Blue white screening of transformants:

When exogenous DNA fragments are inserted into pBLUE-TLater, due to external factorsThe nucleic acid sequence of DNA has altered the coding of the LacZ gene, thereby affecting its productsb-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. sometimesInserting fragments has no impactThe lacZ gene reading frame or insertion fragment is too small, in which 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, and incubate overnight at 37 ℃.
2). Identification of transformants:

a.Extract the plasmid with the above cultured white colony liquid, use ECOR I single enzyme digestion or other appropriate enzyme digestion, and use agarose gel electrophoresis to check the size of the fragment to determine whether it contains the target fragment.

b.Select white bacterial colonies for direct PCR detection (please refer to Molecular Cloning Version 3 or consult us)

c. Use T3 and T7 promoter primers or other suitable primers for sequencing to determine if the target clone is present.

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 ÷ 2.984kb vector] x 3/1=40.2ng

PBLUE-T Vector Cloning Kit T4 Principle Vector and Others