DNA Assembly Plus Kit By Lablead biotech,

Product Description

DNA Assembly Mix Plus KIT CAT：D0204P

Price:
U.S.＄97.00 25T

＄172.00 50T .＄59.00 10T

product ID: D0204P
Storage conditions:-20°C

Component

Component	10T	25T	50T
DNA Assembly Mix PLUS	50μl	125μl	250μl
pUC19 Control Plasmid, Linearized (Ampr, 40 ng/μl)	5μl	5μl	5μl
500 bp Control Fragment (20 ng/μl)	5μl	5μl	5μl

Product introduction:
The seamless cloning technology based on the principle of recombination, as a new generation cloning method, does not rely on the tedious enzyme cutting and linking steps, nor does it need the end complement equal operation. According to the recombination of the DNA fragment and the 15-25nt homologous sequence at the end of the linearized vector, the inserted fragment can be cloned to any site of any linear vector, and the vector self-linking background is very low. It is a simple, fast and efficient DNA directed cloning technique.
The DNA Assembly Mix Plus KIT can complete single fragment recombination in as little as 5 minutes, with a positive rate of over 95%. The co-factors added in Mix can effectively improve the cloning positive rate. The optimized reaction system can tolerate impurities contained in unpurified PCR products to a certain extent. The updated version of seamless cloning kit has higher positive rate and better compatibility.
Simple—compatible PCR and enzyme digestion system, eliminating the tedious purification steps
high efficiency—can stably support 5-6 fragments in the same clone system
Stable and reliable—placed at 37℃for a week or frozen and thawed 30 times, no obvious decrease
Operation method:
1.Summary of experimental process
2. Preparation of linearized cloning vector
Appropriate cloning sites were selected to linearize the vector. The linearization of the vector could be accomplished by enzyme digestion or reverse PCR amplification.
①Preparation by enzyme digestion
Double digestion with LabFD™ rapid endonuclide is recommended to linearize the vector completely to reduce the transformation background (false positive clones). If single enzyme digestion is used for linearization, the enzyme digestion time can be extended appropriately to reduce cyclic plasmid residue.
Note 1: Vectors linearized by double digestion do not require dephosphorylation, while those linearized by single digestion require dephosphorylation.
Note 2: After digestion, the fast endonuclide should be inactivated or purified from the target product before being used in the recombinant reaction.
②Preparation by reverse PCR amplification
To reduce the introduction of amplification mutations, high-fidelity PCR Mix is recommended for amplification. It is recommended to use prelinearized plasmids as templates to reduce the influence of cyclic plasmids on the positive rate of clones.

3. Design of PCR primers for insertion fragments
The 5' end of the PCR primer must contain sequences of 15 to 25 nt (18nt recommended) homologous to the end of its adjacent fragment (insert fragment or vector). If the carrier has a sticky end and the 3' end is protruding, the design of primer must include protruding part. If the 5' end is protruding, the primer design may or may not include the protruding part.
Insert fragment forward amplification primer:
5' -- upstream vector terminal homologous sequence + cleavage site (optional) + gene-specific forward amplification sequence -- 3'
Insert fragment back amplification primer:
3' -- gene-specific backamplified sequence + restriction site (optional) + downstream vector terminal homologous sequence -- 5'

Note 1: The region with no repeat sequence and even GC content was selected for cloning as far as possible. The recombinant efficiency was the highest when GC content was 40%~60% in the 25 nt region upstream and downstream of the vector cloning site.
4. PCR amplification of the inserted fragment
High-fidelity PCR Mix is recommended for amplification to reduce the introduction of amplification mutations. It is recommended to use purified PCR products for seamless cloning. If PCR products are identified as specific amplification products by agarose gel electrophoresis, they can be used directly, but the sample volume should not exceed 20% of the total reaction volume.

component	Reaction system	Negative control	Positive control (if necessary)
DNA Assembly Mix	5μl	5μl	5μl
linearization carrier（ng）	50~200	50~100	pUC19 Control Plasmid,Linearized, 1μl
Insert fragment	10~200ng	-	500 bp Control, Fragment, 1μl
ddH2O	To 10μl

5. Recombination reaction
①The following reaction system is configured in the ice bath:
a. The optimal carrier dosage (ng)=0.02× the number of carrier base pairs, i.e. 0.03pmol.
b. Insert single fragment, the optimal fragment amount (ng) = 0.04× the number of fragment base pairs.
Note 1: If the length of the inserted single fragment is larger than the carrier, the amount of the carrier and the inserted fragment should be exchanged;
Note 2: If the length of the inserted segment is less than 200 bp, 5 times the amount of carrier should be used for the inserted segment;
Note 3: If the dosage calculated according to the above formula exceeds the minimum/maximum value, it is recommended to use the minimum/maximum value directly;
Note 4: The positive rate of cloning will decrease if the vector fragment is too long and the insertion fragment is too long.
After the recombination reaction system is prepared, gently absorb and mix each component with a pipette gun to avoid bubbles and do not swirl.
②The reaction system was placed at 50℃for 5-15min.

Note 1: It is recommended to use PCR instrument with relatively accurate temperature control for reaction. Insufficient reaction time or too long reaction time will reduce cloning efficiency.
Note 2: The recommended reaction time is 5-15min when one to two fragments are inserted, and 30-60min when three to five fragments are inserted.
Note 3: When the carrier skeleton is more than 10 kb or the insertion fragment is more than 4 kb, it is recommended to extend the reaction time to 15-30min.
Note 4:50℃After the reaction is completed, it is recommended to perform instantaneous centrifugation and collect the reaction liquid to the bottom of the tube.
③The reaction liquid centrifuge tube was placed on ice to cool, and then transformed or stored at -20℃.
Note 1: The recombinant product stored at -20℃is recommended to be used within 1 week.

6. Transformation of recombinant products
Take 5-10 μl reaction solution, add it into 100μl receptive cells, slowly suck and mix, and place it on ice for 30min. Heat shock 45~60sec at 42℃, ice bath 5 min. Add 500 μl SOC or LB medium and shake culture at 37℃for 40 to 60min (200 rpm). The bacterial solution was evenly coated on a plate containing the corresponding antibiotic, and was placed at 37℃overnight for culture.
Note 1: The final positive rate of clones varies with different receptive cells, and it is recommended to use receptive cells with conversion efficiency >108 CFU/μg.
Note 2: The number of colonies depends on the number and purity of PCR products and linearized vectors;
Note 3: Positive control plates usually grow a large number of single white colonies, while negative control plates grow very few colonies.

7. Positive clone detection
Single colonies were selected and mixed in 10 μl ddH2O, cleaved at 95℃for 10 min, then 1 μl lysate was used as a template for colony PCR identification, or single colonies were inoculated into resistant medium for overnight culture, then plasmid was extracted for enzyme digestion identification.
Note 1: It is recommended to use at least one universal primer for colony PCR to avoid false positive results.
Note 2: The positive results can be further identified by sequencing if necessary.

Country:	China
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Place of Origin:	China
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Delivery Time:	7-15 days if in stock
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Product Group :	Molecular Biology