Why Johnny Can’t clone: Common Pitfalls and Not So Common Solutions

Matsumura, Ichiro

doi:10.2144/000114324

Cited by 14 publications

(13 citation statements)

References 77 publications

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“…The approach described here builds upon the following classical cloning techniques (18), except as noted. A more detailed step-by-step protocol is included in the Supplementary Material.…”

Section: Methodsmentioning

confidence: 99%

Semi-Automated Tip Snip Cloning of Restriction Fragments into and out of Plasmid Polylinkers

Matsumura

2017

BioTechniques

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Synthetic biologists rely on semi-synthetic recombinant plasmids, but DNA synthesis is constrained by practical limits on length, accuracy, and sequence composition. Cloned DNA parts can be assembled into longer constructs via subcloning, but conventional methods are labor-intensive. One-pot recombination reactions are more convenient but harder to troubleshoot, and those that depend on PCR to create fragments with compatible ends necessitate re-sequencing. The Tip Snip protocol described here enables the subcloning of an insert from one plasmid polylinker into another without PCR or gel purification steps. Cohesive ends of unwanted restriction fragments are snipped off by additional restriction endonucleases. The resulting short fragments (snippets) are eliminated by hybridization to complementary oligonucleotides (anti-snippets) and subsequent size-selection spin-column chromatography. Unwanted linear donor vectors are ligated to double-stranded oligonucleotides (unlinkers) so that only the desired insert and recipient plasmid form circular DNA capable of transforming bacteria. This new method is compatible with high-throughput processing and automated liquid handling, and because no specialized vectors, reagents, selection schemes, or analytical techniques are required, the barriers to adoption are low.

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“…The approach described here builds upon the following classical cloning techniques (18), except as noted. A more detailed step-by-step protocol is included in the Supplementary Material.…”

Section: Methodsmentioning

confidence: 99%

Semi-Automated Tip Snip Cloning of Restriction Fragments into and out of Plasmid Polylinkers

Matsumura

2017

BioTechniques

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“…T4 DNA Ligase efficiently ligates sticky ends of both DNA target and vector DNA created by EcoR1 because of the presence of overhangs on both ends of the fragments. Ligation process depends much on the concentration of reactants, catalysts, and other chemicals present in the mixture, only to a lesser degree on temperature and time (Matsumura 2015). The ligation proceeded at a temperature of 4 0 C for one night to increase the stability of hydrogen bond that connected the nucleotides chain in ligation process.…”

Section: Ligation Of Dna Fragment With Vectormentioning

confidence: 99%

“…The efficiency of bacteria cells to uptake the recombinant plasmids determines the success of a cloning project (Matsumura 2015). Among two transformation methods available, i.e.…”

Section: Cloning and Recombinant Bacteria Selectionmentioning

confidence: 99%

“…Plasmid DNA digestion with restriction enzyme Matsumura (2015) suggested that every stage in cloning experiment should be confirmed by electrophoresis in an agarose gel. Before the target DNA was transformed into a bacteria cell, the target DNA was digested with a restriction enzyme.…”

Section: Plasmid Isolation and Pcr Plasmidmentioning

confidence: 99%

“…Thus, effective and efficient cloning protocol is highly needed to obtain the optimum result of genomic library construction. Although molecular cloning is very basic molecular work, and most laboratories do not have problems conducting the experiment, many others still difficult to develop an optimum protocol (Matsumura 2015). Cloning protocol for genomic library construction conventionally starts from DNA isolation, DNA digestion using restriction enzyme to create DNA fragments, ligation of DNA fragments to a vector, transformation of recombinant vector to bacteria cell, multiplication of bacteria cell into colonies, plasmid vector isolation, and DNA sequencing of insert.…”

Section: Introductionmentioning

confidence: 99%

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Development of protocols for genomic library construction of Agarwood (Aquilaria malaccensis)

Siregar

Suharsono

2017

Biodiversitas

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Abstract. Siregar UJ, Maulana MI, Suharsono UW. 2017. Development of protocols for genomic library construction of Agarwood (Aquilaria malaccensis). Biodiversitas 18: 1150Biodiversitas 18: -1158. Agarwood is one of non timber forest products (NTFP) which has high economic value. Diminishing agarwood trees in natural forest due to intensive harvesting has shifted the production paradigm to forest plantation. In order to support agarwood tree improvement, investigation on the agarwood tree genomes and genes involved in agarwood production is highly needed through genomic library construction. This research aimed at establishing cloning protocols for genomic library construction of agarwood tree (Aquilaria malaccensis) until sequencing process. Genomic DNA was isolated using DNeasy Plant Mini Kit from Qiagen, then was digested with EcoR1, and was ligated to pGem®-T Easy vector system before it was finally transformed and was cloned to E.coli strain DH5α. Confirmation of DNA inserts was done using PCR colony with universal primer of SP6 and T7, plasmid isolation and also PCR plasmid and plasmid DNA digestion used Quick Plasmid Miniprep Kit from Thermofisher. Cloned A. malaccensis DNA fragments were sequenced and BLAST at NCBI site. The cloning successfully obtained five white colonies which contain inserted A. malaccensis DNA fragments with the size of 136-225 bp. PCR colony, plasmid isolation, PCR plasmid, and plasmid DNA digestion had confirmed the existence of inserted A. malaccensis DNA genome inside the bacteria cells. Sequencing and BLAST showed that DNA inserts from colony number 6, 8, and 9 were not similar with A. malccensis sequence that was recorded in NCBI, indicating that the genomic region in this library construction is different from the genomic DNA sequences of A. malaccensis in NCBI.

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The immune system of prokaryotes: potential applications and implications for gene editing

Liu,

Wang

et al. 2024

Biotechnology Journal

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Gene therapy has revolutionized the treatment of genetic diseases. Spearheading this revolution are sophisticated genome editing methods such as TALENs, ZFNs, and CRISPR‐Cas, which trace their origins back to prokaryotic immune systems. Prokaryotes have developed various antiviral defense systems to combat viral attacks and the invasion of genetic elements. The comprehension of these defense mechanisms has paved the way for the development of indispensable tools in molecular biology. Among them, restriction endonuclease originates from the innate immune system of bacteria. The CRISPR‐Cas system, a widely applied genome editing technology, is derived from the prokaryotic adaptive immune system. Single‐base editing is a precise editing tool based on CRISPR‐Cas system that involves deamination of target base. It is worth noting that prokaryotes possess deamination enzymes as part of their defense arsenal over foreign genetic material. Furthermore, prokaryotic Argonauts (pAgo) proteins, also function in anti‐phage defense, play an important role in complementing the CRISPR‐Cas system by addressing certain limitations it may have. Recent studies have also shed light on the significance of Retron, a reverse transcription transposon previously showed potential in genome editing, has also come to light in the realm of prokaryotic immunity. These noteworthy findings highlight the importance of studying prokaryotic immune system for advancing genome editing techniques. Here, both the origin of prokaryotic immunity underlying aforementioned genome editing tools, and potential applications of deaminase, pAgo protein and reverse transcriptase in genome editing among prokaryotes were introduced, thus emphasizing the fundamental mechanism and significance of prokaryotic immunity.

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Why Johnny Can’t clone: Common Pitfalls and Not So Common Solutions

Cited by 14 publications

References 77 publications

Semi-Automated Tip Snip Cloning of Restriction Fragments into and out of Plasmid Polylinkers

Semi-Automated Tip Snip Cloning of Restriction Fragments into and out of Plasmid Polylinkers

Development of protocols for genomic library construction of Agarwood (Aquilaria malaccensis)

The immune system of prokaryotes: potential applications and implications for gene editing

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