One Step Seamless Cloning Kit

One Step Seamless Cloning Kit (Single segment)

One Step Seamless Cloning Kit

Catalog Number:42111

Packaging quantity:

Product storage:-20°C storage, avoid repeated freezing and thawing

Product Description:The one-step seamless cloning kit does not rely on T4 ligase and is not limited by the cleavage sites of the vector and target fragment. Instead, it directly uses the overlapping fragment recombination method. By using a special enzyme combination, any linearized vector can be directed and recombined with PCR fragments that have 16-25bp overlapping regions at both ends. It can efficiently and seamlessly clone A-terminal or flat terminal PCR product fragments to any site of the vector within 30 minutes.

Product Features:

1. Within 30 minutes, a 50bp-15kb PCR amplification fragment (flat/A-terminus) can be cloned and inserted into any position of any vector.

2. Cloning can be performed at any site without being limited by the availability of the vector and insertion site, as well as the flat/sticky ends.

3. Seamless cloning, insertion points will not introduce unwanted base sequences.

4. Not dependent on ligases and phosphatases, efficient, accurate, with a cloning positivity rate of over 95%.

Schematic diagram of the principle of one-step seamless cloning kit:

Preparation of linearized vectors and inserted DNA fragments:

APreparation of Linearized Carrier

(1). Enzyme digestion source: Linear carrier obtained by enzyme digestion, with flat or sticky ends, single or double enzyme digestion, and gel recovery after enzyme digestion.

Note: There is no double stranded DNA ligase in the one-step seamless cloning reaction system, and there will be no carrier self ligation reaction. Therefore, even linearized carriers prepared by single enzyme digestion do not require terminal dephosphorylation treatment. The false positive clones (without inserted fragments) that appear after the transformation of recombinant products are formed by the conversion of non linearized circular vectors that cannot be digested by enzymes. We recommend enzyme digestion followed by gel recovery to reduce the proportion of this non linearized carrier to the lowest possible level.

(2). Reverse PCR source: It is recommended to use high fidelity DNA polymerase for preparation. If the amplification band is single, the vector can be obtained by purifying the PCR product (item number: DC012-50). Otherwise, the vector can be obtained by gel recovery (item number: DC011-50).

Note: The plasmid template for reverse PCR is also a non-linear vector, which may lead to false positive clones (without inserted fragments). Therefore, digesting the plasmid template with Dpn I endonuclease before purifying the PCR derived linear vector (PCR product) can reduce background and increase positivity rate. However, in general, gel recovery is sufficient to reduce the proportion of non linearized carriers to the lowest possible level. Therefore, we also recommend gel recovery for linearized carriers derived from reverse PCR.

BPreparation of Inserted DNA Fragments

(1). The general principle of one-step seamless cloning primer design is to introduce a linearized cloning vector end homologous sequence at the 5 'end of the primer, so that the amplified products 5' and 3 'of the inserted fragment have sequences (16 bp~20 bp) that are identical to the corresponding ends of the linearized cloning vector, respectively.

(2). Insertion fragment primer design: Cloning primers include insertion fragment specific primer sequences and overlapping sequences.

Cloning forward primers (5 '-3'): linear vector forward 16-25 nt overlap region sequence (counted from the 3 'end)+insertion fragment forward specific primer sequence (18-25 nt)

Cloning Reverse Primer (5 '-3'): Linear Vector Reverse 16-25 nt Overlap Region Sequence (from 3 'End)+Insertion Fragment Reverse Specific Primer Sequence (18-25 nt)

Attention: The base number of the overlapping region should be at least 16 bp, and the Tm value between multiple overlapping regions should be consistent and>60 ° C (AT pair=2 ° C and GC pair=4 ° C). Otherwise, the base number can be extended until it meets the requirements.

According to the structure of the linear carrier end (5 'protrusion, 3' protrusion, flat end), primer design can also be divided into three situations, as shown below:

The two ends of the linearized vector can be any combination of the three terminal structures mentioned above due to different linear methods (such as single enzyme digestion, double enzyme digestion, reverse PCR). The principle of designing insert fragment specific primers should follow the general principles of primer design.

When calculating the annealing temperature of amplification primers, only the Tm value of the gene specific amplification sequence needs to be calculated, and the homologous sequence at the end of the vector should not be included in the calculation.

(3). Enzyme selection: It is recommended to use high fidelity DNA polymerase or PCR Mix (item number: P517-05 or P814-05).

(4). Reaction conditions: Generally, follow the instructions for the specific polymerase used.

(5). Purification insertion fragment

l Optional: If the fragment is derived from a plasmid template and the plasmid has the same resistance as the recombinant vector, digesting the plasmid template with Dpn I endonuclease before purification can reduce the background and increase the positivity rate.

l If the fragment is single, it is recommended to purify the fragment using PCR product purification kit (item number: DC012-100).

l If there is non-specific amplification, it is recommended to use a gel recovery kit (item number: DC011-100) to recover the fragment.

l When using this method for cloning, the enzyme cleavage sites used to linearize the vector may be missing during splicing. If there are strict requirements for the enzyme cleavage sites, it is recommended to pay attention to the selection of enzyme cleavage sites, and if necessary, they can be addedBetween the overlapping region sequence of forward and reverse cloning primers and the specific gene sequenceAdd missing bases to restore the original enzyme cleavage site (see examples below).

l If recombinant plasmids are used for protein expression, attention should be paid to ensuring that the reading frames, protein expression, and purification sequences (such as promoters, RBS sequences, start codons, stop codons, protein tags, etc.) are not disrupted during primer design.

Example of forward primer design (EcoR I enzyme cleavage):

As shown in the figure above, the carrier was cleaved by Ecor I enzyme to form a 5 'protruding end:

According to the above design principles, starting from the 3 'end, calculate back 16bp-25bp (this example uses a 20 bp end overlapping sequence), and add it before the target fragment specific primer sequence. Ensure that the Tm value of the overlapping area remains consistent and>60 ° C (AT pair=2 ° C and GC pair=4 ° C).

The specific forward primers are as follows: 5 'GAGCCTAGGTGAGTTGGCCGNNNNNNNNNNNNNNNNNNNN 3'

Note: After the above primer designs are cloned and connected, the EcoR I enzyme cleavage site will disappear (without retaining the cleavage site).

If it is necessary to preserve the EcoR I cleavage site, the missing EcoR I recognition site sequence aattc needs to be filled in between the 20 bp overlapping sequence at the end of the vector and the target fragment specific primer sequence. After cloning and ligation, the EcoR I cleavage site still exists (the cleavage site is preserved).

Specifically, as follows: 5 'GAGCCTAGGTGAGTTGGCCGaattc NNNNNNNNNNNNNNNNNNNN 3'

Example of reverse primer design (Hind III enzyme cleavage):

As shown in the figure above, the carrier is cleaved by Hind III enzyme to form a 5 'protruding end:

According to the above design principles, starting from the 3 'end, calculate back 16bp-25bp (this example uses a 20 bp end overlapping sequence), and add it before the target fragment specific primer sequence. Ensure that the Tm value of the overlapping area remains consistent and>60 ° C (AT pair=2 ° C and GC pair=4 ° C).

The specific reverse primers are as follows: 5 'CTGCCATCGGATCGTTCGCANNNNNNNNNNNNNNNNNNNN 3'

Note: After the above primer designs are cloned and connected, the Hind III cleavage site will disappear (without retaining the enzyme cleavage site).

If it is necessary to preserve the Hind III cleavage site, the missing Hind III recognition site sequence agctt needs to be filled in between the 20 bp overlap sequence at the end of the vector and the target fragment specific primer sequence. The Hind III cleavage site still exists (the cleavage site is preserved).

Specifically, as follows: 5 'CTGCCATCGGATCGTTCGCAagctt NNNNNNNNNNNNNNNNNNNN 3'

Operation steps for seamless cloning reaction:

Attention: 2 x OneStep Cloning Mix contains a connection enhancer PEG, which is very viscous. When taken out of the refrigerator at low temperatures, it becomes even more viscous. You can freeze it in your palm for a few minutes and increase the temperature to reduce the viscosity (without affecting the quality). Gently mix and centrifuge to collect it at the bottom of the tube in an instant.

1. Establish a reaction system according to the table below (PCR tubes can be used to prepare at room temperature)

*The carrier is generally used at 20-50 ng, and the optimal molar ratio of the inserted fragment to the carrier is between 2:1-3:1; If the inserted fragment is less than 200bp, the molar ratio of the inserted fragment to the carrier is 5:1.

2. Gently mix well, at 50°C reaction takes 15 minutes (can be performed on a PCR instrument). After the reaction is complete, place the PCR tube on ice and directly convert or store it in -20 ° C°C. Short fragments such as 100bp-1kb can obtain sufficient transformants in just 15 minutes, while longer fragments can be linked to extend the reaction time to 60 minutes.

3. Take 5 μ l of the reaction product and convert it according to the instructions of the competent cells (if there are few transformants, all the products can be converted and the conversion solution can be coated on a plate).

Identification of positive clones:

Depending on the specific situation, colony PCR identification and plasmid extraction (item number 31013-100) can be selected for restriction enzyme identification or sequencing identification.

One Step Seamless Cloning Kit