One Step DNA Cloning Kit

Composition

Storage

Store at -20℃ with an expiration date of 2 years.

Introduction                                                                                        

This product is a seamless cloning technology based on the principle of gene recombination, which can achieve directional cloning of DNA simply, quickly and efficiently. Instead of relying on cumbersome digestion, ligation steps, and end complementing operations, the insert gene can be recombined to any site of any linear vector by recombining the DNA fragment with the 15~25 nt homologous sequence at the end of the linearized vector.

This product can complete the recombination of multiple DNA fragments at one time, and the fastest recombination of a single fragment is only 5 min, and the positive rate is higher than 95%. The optimized reaction system can effectively improve the positive rate of clones and can tolerate impurities in unpurified PCR products to a certain extent, with higher positive rate and stronger compatibility.

Protocol

1.     Linearized vectors preparation

Select the appropriate cloning site and linearize the vector, either by digestion or reverse PCR amplification.

(1) Enzyme digestion preparation

It is recommended to use a fast endonuclease (single or double digestion) to linearize the vector to reduce transformation background and reduce false positive cloning from undigested vector transformation. When using common endonuclease single digestion, the digestion time can be appropriately extended to reduce the residue of the circular plasmid.

* Dephosphorylation is required for linearized vectors for single digestion and no dephosphorylation is required for double digestion.

* After digestion is completed, it is recommended to inactivate the endonuclease or purify the target product for recombination reaction.

(2) Reverse PCR amplification

High-fidelity PCR Mix is recommended for amplification to reduce the probability of amplifying mutations. It is recommended to use the linearized plasmid as a template to reduce the effect of circular plasmid template carryover on the cloning positivity rate.

* When using a circular plasmid as a template, the PCR product needs to be digested with MinuteCut™ Dpn I. (Cat. No. 6020050) and then purified and recovered to reduce the effect of residual circular plasmid template on the cloning positivity rate.

* For multi-fragment cloning, it is recommended to use the PCR product after purification.

2.     Insert preparation

(1) Primer design principles

The primers of the insert include two parts, the overlap region of 15-25 nt and the specific primer, that is, the 5' end of the positive/reverse primer of the insert is introduced with 15-25 nt (18 nt recommended) homologous to the end of the adjacent fragment (insert or linearized vector), so that the end of the amplified insert has the same homology sequence as the end of the adjacent fragment.

F forward primer (5'-3'): upstream vector ends homologous sequence + enzyme cleavage site (optional) + gene-specific forward amplification sequence

R reverse primer (5'-3'): downstream vector ends homologous sequence + enzyme cleavage site (optional) + gene-specific reverse amplification sequence

* If the vector is sticky with a sticky end and the 3' end is protruding, the primer design must include the protrusion, and if the 5' end is protruding, the primer design may or may not contain the protrusion.

* Try to choose the region with no repeats and uniform GC content for cloning, and the recombination efficiency is the highest when the GC content is 40%~60% in the 25 nt region upstream and downstream of the vector cloning site.

* When calculating the Tm value of amplification primers, only the Tm value of the specific primer needs to be calculated, and the additional sequences introduced do not need to be calculated.

(2) PCR amplification of inserts

High-fidelity PCR Mix is recommended for amplification to reduce the introduction of amplification mutations. Seamless cloning of purified PCR products is recommended, and can be used directly if the PCR product is identified by agarose gel electrophoresis as a specific amplification product, but the dosing volume should not exceed 20% of the total reaction volume.

3.     Recombinant reactions

(1)      Place the following reaction systems in an ice water bath.

*1 Optimal vector dosage (ng) = 0.02 × vector base pairs, i.e., 0.03 pmol.

*2 Insert one fragment, optimal fragment dosage (ng) = 0.04 × fragment base pair; Insert multiple fragments, optimal amount per fragment (ng) = 0.02 × fragment base pairs.

* If the length of the insert is larger than the vector, the vector and insert dosage should be swapped.

* If the length of the insert is less than 200 bp, the insert should be used with 5 times the vector dosage.

* If the dosage calculated according to the above formula exceeds the min/max value, it is recommended to use the min/max dosage directly.

* If the vector fragment is too long, the insert is too long, or the number of fragments is too high, the cloning positive rate will be reduced. After the recombinant reaction system is prepared, use a pipette to gently mix the components to avoid bubbles and do not vortex.

* The dosage of each component should not be less than 1 μl, and the components can be diluted before adding.

(2)      Place the reaction system at 50°C for reaction and follow the reaction time.

* When the vector fragment > 10 kb or the insert > 4 kb, it is recommended to extend the reaction time to 30~60 min.

* It is recommended to use a PCR instrument with more accurate temperature control for the reaction, as the cloning efficiency will be reduced if the reaction time is insufficient or too long.

* After the 50°C reaction is completed, it is recommended to spin down to collect the reaction solution to the bottom of the tube.

(3)      Cool the recombinant product on ice and then transform or store at -20°C for later use.

* Recombinant products stored at -20℃, it is recommended to use within 1 week.

4.     Transformation of recombinant products

(1)Take 5~10 μl of the reaction solution for transformation, and follow the instructions of the competent cell instructions, or follow the following steps.

(2)Take 5~10 μl of the reaction solution, add it to 100 μl of competent cells, flick the tube wall and mix well, and take an ice bath for 30 min.

(3)Heat shock at 42℃ for 45~60 sec, leave on ice for 3 min.

(4)Add 900 μl SOC or LB culture medium (without antibiotics), incubate at 37°C for 60 min (160-220 rpm).

(5)Pre-warm the corresponding resistant LB solid medium plates in a 37℃ incubator.

(6)Centrifuge at 5,000 rpm (2,500×g) for 4 min and discard 900 μl of supernatant. Gently pipette to resuspend the bacteria with the remaining medium and gently coat the pre-warmed plate with a sterile coating stick.

(7)Plate until the liquid is absorbed, then incubate inversion in a 37℃ incubator overnight.

5.     Positive clone test

(1)Colony PCR: Pick a monoclone to 10 μl ddH₂O and mix well as a template; Use appropriate forward and reverse primers for colony PCR identification.

* At least one primer sequence should be on the vector, not designed for recombination.

(2)Enzyme digestion method: single colonies were picked into a suitable resistant liquid medium and cultured overnight, and the plasmid was extracted for enzymatic identification.

(3)Sequencing identification: Sequencing analysis is performed using appropriate primers on the vector.