Ultra One Step Cloning Kit

1. Prepare the target fragment

(1) Enzyme selection: The insert fragment can be amplified using any PCR enzyme, such as Taq enzyme or high-fidelity enzyme, regardless of whether the product has an A tail at the end (it will be removed during the recombination process and will not appear in the final vector). To reduce the introduction of mutations, it is recommended to use a high-fidelity enzyme to amplify the fragment.

(2) Single fragment homologous recombination

a. Primer design principle: The end sequences on both sides of the insertion site on the linearized vector are introduced at the 5' end of the forward/reverse amplification primer of the insert fragment, i.e., the homology arms (15-25bp, excluding the restriction site).

b. Forward primer:

5'-upstream end sequence of vector (15-25bp) + restriction site (can be retained or deleted) + gene-specific forward amplification primer sequence-3'

c. Reverse primer:

5'-downstream end sequence of vector (15-25bp) + restriction site (can be retained or deleted) + gene-specific reverse amplification primer sequence-3

(3) Multi-fragment homologous recombination

a. Primer design principle: Introduce homologous sequence at the 5' end of the primer so that multiple inserted fragments and the first and last inserted fragments and the linearized vector have completely consistent sequences (15-25bp, excluding restriction site) that can homologously recombine with each other.

b. Forward amplification primer for the most upstream fragment:

5'-upstream end sequence of the vector (15-25bp) + restriction site (can be retained or deleted) + gene-specific forward amplification primer sequence-3'

c. Reverse amplification primer for the most downstream fragment:

5'-downstream end sequence of the vector (15-25bp) + restriction site (can be retained or deleted) + gene-specific reverse amplification primer sequence-3'

d. The homology arms between the middle inserted fragments can be taken from the 3' end of the previous sequence 15-25bp added to the 5' end of the next sequence, or the 5' end of the next fragment 15-25bp added to the 3' end of the previous sequence, or a part of each of the two fragments can be taken as a homology sequence, totaling 15-25bp, and added to the end of the other fragment respectively.

2. Prepare linearized vector: Select a suitable insertion site and linearize the vector by restriction endonuclease or reverse PCR amplification.

Note:

(1) Regarding the selection of insertion site: Try to select a position without repetitive sequences and with a GC content of 40%-60% in the 15-25bp region upstream and downstream of the vector cloning site to insert the target fragment.

(2) If restriction endonuclease digestion is used to prepare linearized vector, it is recommended to use double digestion, followed by single digestion linearization, and appropriately extend the digestion time to reduce the generation of false positives.

(3) When preparing linearized vector by reverse PCR amplification, it is recommended to use a high-fidelity polymerase to reduce the introduction of mutations. It is recommended to use 0.1-1ng plasmid template in the PCR system, or use a pre-linearized plasmid as a template to reduce false positives caused by residual circular plasmid template.

3. Recombination and transformation

(1) Concentration determination (purified):

It is recommended to use instruments based on absorbance such as NanoDrop, Onedrop, Qubit, PicoGreen, etc. to determine the concentration of the target fragment and the linearized vector. The A260/A280 value should be between 1.8-2.0.

(2) Amount of fragments and vectors:

a. For single-fragment homologous recombination, the molar ratio of vector to insert is 1:2, the recommended amount of vector is 0.03pmol, the recommended amount of insert is 0.06pmol, and the actual amount of insert should be greater than 20ng. The amount can be adjusted according to actual conditions.

Note: If the length of the insert is greater than the length of the vector, the amount of fragment and vector should be interchanged, and the insert should be calculated as the vector and the vector as the insert.

b. For multi-fragment homologous recombination, the actual amount of each insert should be greater than 10ng, and the molar ratio of vector to each insert is 1:1. The amount can be adjusted according to actual conditions.

Note: When the optimal amount calculated by the following formula is less than 10ng, 10ng can be used directly.

c. For single-fragment homologous recombination, if the PCR product has no nonspecific amplification bands, it can be used directly without DNA purification. The total volume added should not exceed 1/5 of the reaction system volume, i.e. 2μl, but the recombination efficiency will be reduced (recombination after purification is recommended).

d. This protocol provides a rough calculation method for the relationship between the molar number and the DNA mass:

For single-fragment homologous recombination reaction:

Recommended amount of vector = [0.02×number of base pairs of cloning vector] ng (0.03pmol)

Recommended amount of fragment = [0.04×number of base pairs of inserted fragment] ng (0.06pmol)

For multi-fragment homologous recombination reaction:

Recommended amount of vector = [0.02×number of base pairs of cloning vector] ng (0.03pmol)

Recommended amount of single fragment = [0.02×number of base pairs of each fragment] ng (0.03pmol)

(3) Prepare the reaction system (operate on ice):

Note:

a. X and Y are the amounts of each insert and vector calculated according to the formula. It is recommended that the actual amount of each component added should not be less than 1μl to ensure the accuracy of the sample addition.

b. If the total amount of X and Y added is greater than 5μL, it is recommended to expand the reaction system, such as 20μl, and increase the amount of Cloning Mix.

c. The positive control is used to exclude the influence of other experimental materials and operating factors; the negative control is used to confirm whether there are circular plasmid residues in the fragment and linearized vector.

(4) Reaction conditions:

a. Single fragment recombination reaction: 50℃, 5 min;

b. 2-3 fragment recombination reaction: 50℃, 15 min;

c. 4-5 fragment recombination reaction: 50℃, 30 min;

After the reaction is completed, it needs to be transferred to 4℃ or immediately placed on ice for cooling.

Note:

a. It is recommended to perform the reaction on an instrument with relatively precise temperature control such as a PCR instrument.

b. The reaction time is recommended not to exceed the recommended time.

c. After the recombination is completed, the product can be transformed immediately, or it can be stored at -20℃ for one week and thawed and transformed when needed.

4. Transformation of recombinant products

(1) Take the competent cells out of the -80℃ freezer and thaw them on ice.

(2) In a clean bench, take 5-10μl of the recombinant product and slowly add it to 100μl of the competent cells. Gently tap the tube wall to mix (do not oscillate to mix), and let it stand on ice for 30 minutes.

Note: The volume of the recombinant product transformation is recommended not to exceed 1/10 of the volume of the competent cells used.

(3) After heat shock in a 42℃ water bath for 30 seconds, immediately place it on ice to cool for 2-3 minutes.

(4) Add 900μl of SOC or LB liquid culture medium without antibiotics and shake the culture at 37℃ for 1 hour (200-220r/min).

(5) Centrifuge the bacterial solution at 5000r/min (2500g) for 3 minutes, discard 900μl of the supernatant, and resuspend the bacteria with the remaining culture medium. Use a sterile spreading rod to evenly spread the resuspended bacterial solution onto a plate containing the corresponding resistance, and invert and culture it in a 37℃ incubator overnight (12-16h).

5. Screening of positive clones

(1) Colony PCR method: Pick a single clone and mix it in 10μl ddH₂O as a template, and use appropriate forward and reverse primers for colony PCR identification. Use at least one primer on the vector as the amplification primer.

(2) Enzyme digestion method: Pick a single clone and culture it in a liquid culture medium with appropriate resistance overnight, extract the plasmid for enzyme digestion identification.

(3) Sequencing identification: Pick a single clone and culture it in 200-500μL liquid culture medium with appropriate resistance, shake the bacteria at 37℃ for 1-3h (200-220r/min), and use the appropriate primers on the vector for sequencing analysis; you can also extract the plasmid and perform plasmid sequencing.

Notes:

1. Primer design is very important. The length of the homology arm should be 15-25bp (excluding the enzyme cleavage site), and the GC content should be 40%-60%.

2. The amount of linearized vector and insert fragment should not be too little or too much, which will lead to low recombination efficiency or low positive rate. It is recommended to refer to the recommended amount.

3. The volume of unpurified DNA should not exceed 1/5 of the volume of the reaction system. If the recombination effect is not ideal, it is recommended to purify it before the ligation reaction.

4. Low efficiency of competent cells may result in few clones on the plate. It is recommended to verify the stored competent cells from time to time to ensure that the transformation efficiency is >10⁸cfu/μg.