Journal
BMC BIOTECHNOLOGY
Volume 17, Issue -, Pages -Publisher
BIOMED CENTRAL LTD
DOI: 10.1186/s12896-017-0346-5
Keywords
Restriction-free cloning; Gene reconstitution; Single-primer PCR; High-throughput cloning
Categories
Funding
- Hebei Agricultural University [ZD201622]
- National Natural Science Foundation of China [31271997, 31301616]
Ask authors/readers for more resources
Background: Restriction-free (RF) cloning, a PCR-based method for the creation of custom DNA plasmids, allows for the insertion of any sequence into any plasmid vector at any desired position, independent of restriction sites and/ or ligation. Here, we describe a simple and fast method for performing gene reconstitution by modified RF cloning. Results: Double-stranded inserts and acceptors were first amplified by regular PCR. The amplified fragments were then used as the templates in two separate linear amplification reactions containing either forward or reverse primer to generate two single-strand reverse-complement counterparts, which could anneal to each other. The annealed inserts and acceptors with 5' and 3' cohesive ends were sealed by ligation reaction. Using this method, we made 46 constructs containing insertions of up to 20 kb. The average cloning efficiency was higher than 85%, as confirmed by colony PCR and sequencing of the inserts. Conclusions: Our method provides an alternative cloning method capable of inserting any DNA fragment of up to at least 20 kb into a plasmid, with high efficiency. This new method does not require restriction sites or alterations of the plasmid or the gene of interest, or additional treatments. The simplicity of both primer design and the procedure itself makes the method suitable for high-throughput cloning and structural genomics.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available