ClonPlus One Step Cloning Mix
Fast, seamless targeted cloning in as little as 5 minutes; broad compatibility and high positive rate.
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Instruction manual- Product Description
- Composition
- Precautions
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Product Overview
ClonPlus employs a simple, rapid, and highly efficient technique for the directional cloning of DNA fragments, enabling the targeted insertion of a desired fragment into any site of any vector. The vector is first linearized, and the 5′ ends of the two PCR primers used to amplify the target fragment are each engineered to include sequences complementary to the linearized vector’s termini. As a result, following PCR amplification, the 5′ and 3′ ends of the target fragment each bear 15–20 nucleotides of sequence homologous to the corresponding ends of the linearized vector. By mixing the PCR product—bearing these homologous sequences at both ends—with the linearized vector in a defined ratio, and under the catalysis of recombinase, directional cloning of the DNA fragment can be achieved in as little as 5–45 minutes.
ClonPlus One Step Cloning Mix is a seamless cloning kit that efficiently performs homologous recombination of single or multiple DNA fragments in a single reaction, offering broad compatibility and a high positive rate.
Storage conditions
This product should be stored at −20°C, and repeated freeze–thaw cycles should be avoided as much as possible during use.
Instructions for Use
I. Overview of the Experimental Procedure
II. Preparation of Linearized Carriers
Select an appropriate cloning site to linearize the vector. Whenever possible, choose a site that lacks repetitive sequences and has a GC content of 40%–60% within the 25 bp flanking the cloning site. Vector linearization can be achieved by restriction digestion or inverse PCR; restriction digestion is recommended for its simplicity and speed.
1. Restriction enzyme digestion
Either single‑enzyme or double‑enzyme digestion is acceptable; we recommend using double‑enzyme digestion to linearize the vector. You may extend the digestion time as needed to ensure complete linearization, thereby minimizing residual circular plasmid and reducing background during transformation (false‑positive clones).
2. Reverse PCR amplification
When preparing linearized vectors via inverse PCR, it is recommended to use a high-fidelity DNA polymerase for vector amplification to minimize the introduction of base substitutions during the amplification process. It is also advisable to use a pre‑linearized plasmid as the template to reduce the impact of residual circular plasmid templates on the clone positivity rate. If a circular plasmid is used as the template, the PCR product should be digested with DpnI to cleave any remaining circular plasmid before purification and recovery.
III. Acquisition of the Target Fragment
It is recommended to use a high-fidelity DNA polymerase for amplifying the target fragment to minimize the introduction of base mutations during PCR. Prior to performing homologous recombination, it is advisable to purify the PCR products. After linearizing the vector by restriction digestion, both ends of the vector will be cohesive ends, each protruding by several nucleotides. When designing primers, these overhanging bases may or may not be incorporated into the primers, and the entire restriction site may likewise be included or excluded from the primers.
1. Primer design for a single fragment
Forward primer: 15–20 nt of the vector’s upstream sense strand + 18–25 nt of the target fragment’s upstream sense strand (5′→3′)
Reverse primer: a 18–25 nt complementary sequence to the downstream end of the target fragment, plus a 15–20 nt complementary sequence to the downstream end of the vector (3′→5′).
Note: When submitting the reverse primer sequence to a primer supplier for synthesis, please provide the sequence in the 5′→3′ direction.
For example:
Linearized carrier sequence: 5,- GCCAAATCTGCATCG ---------- TGCTAGAGCTGATTG -3,
3,-CGGTTTAGACGTAGC- - - - - - - -- - ACGATCTCGACTAAC -5,
Target fragment sequence: 5,- CATACGCCCATCGGTGGA ---------- TATGGTCACCGACGTTAG -3, 3,-GTATGCGGGTAGCCACCT- - - - - - -- - - ATACCAGTGGCTGCAATC -5,
Forward primer design: 5,- GCCAAATCTGCATCG CATACGCCCATCGGTGGA -3,
Reverse primer design: 3, - ATACCAGTGGCTGCAATC ACGATCTCGACTAAC -5,
2. Primer design for multiple fragments
Forward primer for the left fragment: 15–20 nt of the vector’s upstream end plus the forward strand of the left fragment’s upstream end, spanning 18–25 nt (5′→3′).
Reverse primer for the left fragment: 18–25 nt complementary to the downstream end of the left fragment, plus 15–20 nt complementary to the upstream end of the middle fragment (3′→5′).
Forward primer for the middle fragment: 15–20 nt of the downstream strand at the 5′ end of the left fragment plus 18–25 nt of the upstream strand at the 5′ end of the middle fragment (5′→3′).
Reverse primer for the middle fragment: 18–25 nt of the downstream complementary strand of the middle fragment plus 15–20 nt of the upstream complementary strand of the right-hand fragment (3′→5′).
Forward primer for the right fragment: 15–20 nt of the downstream strand at the middle fragment’s 3′ end plus 18–25 nt of the upstream strand at the right fragment’s 5′ end (5′→3′).
Reverse primer for the right fragment: 18–25 nt complementary to the downstream strand of the right fragment, plus 15–20 nt complementary to the downstream end of the vector (3′→5′).
Note: When submitting the reverse primer sequence to a primer supplier for synthesis, please provide the sequence in the 5′→3′ direction.
For example:
Linearized carrier sequence: 5,- GCCAAATCTGCATCG ---------- TGCTAGAGCTGATTG -3,
3,-CGGTTTAGACGTAGC- - - - - - - -- - ACGATCTCGACTAAC -5 ,
Left-side fragment sequence: 5,- GGCACTTACCCCAATCAT ---------- AAGGCCCTATATCCAATT -3, 3,-CCGTGAATGGGGTTAGTA- - - - - - - -- - TTCCGGGATATAGGTTAA -5,
Intermediate fragment sequence: 5’- TATATGGCCATACGTAAT ---------- TTAACCCATGGTGGACCT -3 ’ 3’-ATATACCGGTATGCATTA- - - - - - - -- - AATTGGGTACCACCTGGA -5’
Right-side fragment sequence: 5’- CATACGCCCATCGGTGGA ---------- TATGGTCACCGACGTTAG -3 ’ 3’-GTATGCGGGTAGCCACCT- - - - - - -- - - ATACCAGTGGCTGCAATC -5 ’ Forward primer design for the left fragment: 5’- GCCAAATCTGCATCG GGCACTTACCCCAATCAT -3 ’
Reverse primer design for the left fragment: 3’ - TTCCGGGATATAGGTTAA ATATACCGGTATGCATTA -5
Product Composition
Component Specifications Item number ClonPlus One Step Cloning Mix 250 μL 500-201A pUC19 Control Vector, Linearized (Amp＋, 40 ng/ μL) 5 μL 500-201B 500 bp Control Fragment (20 ng/ μL) 5 μL 500-201C Frequently Asked Questions and Solutions
1. Only a few clones were observed on the plate.
Use competent cells with a transformation efficiency greater than 10^8 cfu/μg for transformation.
The PCR products of the linearized vector and the target fragment were recovered and purified by gel extraction, and the purified products were dissolved in ddH2O.
2. A large number of clones do not contain the target fragment.
Increase the amount of restriction enzyme used, extend the digestion reaction time, or purify the digested products by gel extraction.
3. A relatively large number of clones contain the incorrect target fragment.
Optimize the PCR reaction system to enhance specificity.
Purify PCR products by gel extraction.
4. Most of the cloned colonies showed no bands upon PCR.
Optimize the PCR reaction system and protocol.
Keywords
GOONIE
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