PMID:25774528 #3
Description
Submitter 1 full name
Patrícia Andreia Ataíde Fernandes
Submitter 1 ORCID
0009-0008-4347-2063
Submitter 1 GitHub username
PatriciaAtaideF
Submitter 2 full name
No response
Submitter 2 ORCID
No response
Submitter 2 GitHub username
No response
Submitter 3 full name
No response
Submitter 3 ORCID
No response
Submitter 3 GitHub username
No response
PMID
PMID:25774528
Title
Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli
Cloning Detail Level
Cloning information present, but not sufficient to reproduce
Plasmids and Alleles
- p3B: pUC19-derived plasmid containing a genome region from S. japonicus.
Cloning Techniques Used
- Gibson Assembly
- Golden Gate
- Gateway Cloning
- CRISPR/Cas9
- Homologous Recombination
- Traditional Restriction Enzyme Cloning
- PCR Cloning
- Overlap Extension
- In-Fusion Cloning
- De-novo DNA synthesis
- Other (please specify in notes)
Cloning Text (if available)
The pUC19 vector was linearized by PCR amplification. In most cases forward and reverse primers were designed to anneal them apart from each other on the pUC19 sequence (GenBank accession number M77789.2). This resulted in amplification of a simplified pUC19 backbone in which the lacZ gene and the multiple cloning sites were deleted (see Fig. 1). PCR amplification of the receiver plasmids was carried out from 5.0 to 10.0 ng (normal protocol) or 0.5 ng (optimized protocol) of template DNA in a 50 μL reaction volume. Insertion fragments derived from the yeast Schizosaccharomyces japonicus var. versatilis were amplified by PCR from about 30–60 ng of genomic DNA per 50 μL reaction volume. Yeast specific primers were designed based on the S. japonicus var. japonicus genome (GenBank accession number AATM00000000.2). The PCR fragments corresponding to the antibiotic resistance cassettes kanMX, zeoMX, natMX, and hphMX were derived from the plasmids pUG6, pUG66, pAG25, and pAG34, respectively (EUROSCARF collection) [18]. The amount of these templates used per reaction volume of 50 μL was 5–10 ng. The sequences of all oligonucleotides used in this study are listed in the supplementary S1 Table. PCRs for amplification of cloning vectors and inserts were carried out using Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instructions. Optimal PCR primer annealing temperatures were chosen according to the oligonucleotide’s specific Tm or were adjusted empirically. We invariably used 30 cycles of amplification. To generate enough material for gel purification, we normally prepared PCR reactions in duplicates or triplicates. When specified in the text, after PCR we depleted the template plasmid by digesting it with 10 U of DpnI (New England Biolabs, Ipswich, MA, USA) for 1 h in the same buffer used for the PCR. Restriction endonucleases PvuII, EcoRI and HindIII were purchased from New England Biolabs (Ipswich, MA, USA) and used according to manufacturer’s instructions.
Cloning Strategies
Missing information (if any)
No response
Present information (if any)
No response
Assumptions
No response
Notes
I tested one of the examples, but in the paper there are more that can be tested. I tried to do the first one to generate the p3B plasmid, but the homologous recombination was not possible to reproduce (no homology found by OpenCloning).