... in plasmid cloning Perhaps the most widely used application of CcdB is GatewayTM cloning, where the ccdB...counterselectable marker in the destination vector. Successful cloning occurs when the ccdB gene is replaced by the gene...plasmid, they were able to create an efficient cloning tool. The plasmid pKSCTMF contains the mazF gene...ve also helped researchers design and optimize cloning methodologies using counterselection. Since their...Learn more about CcdB, the toxic key to efficient cloning Read all Plasmids 101 blog posts Read about knockout...

...More complex 24-fragment assembly Single insert cloning efficiencies were measured based on the acquisition...Antibiotic selection allowed for high throughput cloning efficiency measurements. We performed additional... Ligase, the mainstay of most biotechnological cloning efforts for over 50 years, prefers ligation of ...Applications & Product Development focusing on DNA cloning and assembly, New England Biolabs, Inc.         ...on ligase efficiency Learn about other plasmid cloning techniques Resources on Addgene.org Find basic...basic molecular biology protocols Find plasmid cloning protocols Check out our molecular biology reference...ligase such as T4 DNA ligase. Inserts, either precloned or in amplicon form (shown above) are flanked ...

...‐inducible bacterial expression system pDawn to clone and express newly identified proteins and enzymes...integration vectors (see for example this yeast cloning kit) she intends to make yeast capable of desaturating... to bacteria and plants. Her research involves cloning cry genes from the soil bacterium Bacillus thuringiensis...bacterial expression system such as the pET GFP LIC cloning vector and the pOKD plasmids. The recombinant Cry...

...Multisite Gateway cloning. A desired insert is cloned using traditional restriction enzyme cloning to create ...standard entry clone. ORFs from an ORFeome Gateway library or any PCR-amplified ORF cloned into pDONR221... promoter-less Gateway® entry clones to be used with other entry clones encoding the ORFs of interest:...iterative cloning steps and the joined modules cannot be separated again by the same cloning enzymes. ...integrase system which is the basis for Gateway cloning. The combination of all 11 att pairs in series .... 2015 Feb 19; 22(2):169-74. High efficiency cloning for enzyme screening Dr. David Ackerley’s lab at...bacterial expression vector which gives close to 100% cloning efficiency and gene expression. The pUCXKT vector...

...process of cloning, combining, and testing these different parts can be quite arduous. The cloning process...interest and cloned into provided destination vectors using low-cost, one-pot golden gate cloning that results...compatible with both standard Gateway cloning and the FNLCR Combinatorial Cloning Platform (CCP); the latter of...terminators for easy cloning of genes and promoters. In addition to USER cloning, this toolkit is also... based on the original EasyClone vectors (Jensen et al 2013) allow for cloning of up to two genes with...STARR-seq, randomly sheared genomic libraries are cloned downstream of a minimal promoter sequence. Should...Integration of Design Automation Research lab Moduclar Cloning (CIDAR MoClo) Parts Kit. This Kit consists of a...

...lab at the Broad Institute and was designed and cloned by Neville Sanjana. This fantastic research tool...in pooled libraries containing thousands of pre-cloned gRNAs. DNA aliquots of the GeCKO v2 pooled libraries... Screening Using CRISPR/Cas9. If you need help cloning a gRNA into lentiCRISPR v2, you can take a look...

... regions of a parental antibody (blue, rat) are cloned into a backbone expressing the constant regions...alternative isotype (purple, mouse). Depending on the cloning process, all constant regions, just the heavy chain...from hybridoma antibody sequences that have been cloned into plasmids. Because their sequences are known...the variable regions of a specific antibody are cloned into a plasmid backbone containing all constant...

...lines such as HEK293 cells. 1st generation AdV cloning capacity is limited to 8.2 kb, and in vivo transgene.... 2nd Gen AdVs were designed to have increased cloning capacity, decreased  potential for the generation...deleted in 2nd generation AdVs, thereby increasing cloning capacity to ~12 Kb. The ever popular pAdEasy system... interest for gene therapy due their increased cloning capacity (up to 36 kb!), long-term transgene expression...shuttle vector (in which the transgene of interest is cloned) and pAdEasy™ which contains the adenoviral genes...

...Learn New Plasmid Cloning Techniques Pushing the Limits of DNA Assembly Plasmid Cloning by PCR Resources... recombinant plasmid construction (restriction cloning), adaptor ligation for next generation sequencing... T4 DNA Ligase in applications such as routine cloning or DNA library preparation. However, when a method...Genetic disease detection and DNA amplification using cloned thermostable ligase." Proceedings of the National...PMC50775. 6. Barany, Francis, and David H. Gelfand. "Cloning, overexpression and nucleotide sequence of a thermostable...11. PubMed PMID: 1756968. 7. Lauer, G., et al. "Cloning, nucleotide sequence, and engineered expression...

... a plasmid? Or how much time you have to spend cloning before you can start your experiment? What about...order? Sometimes, it feels like an eternity of cloning, waiting, and repeating before you can finally ...and money across the board. Consider alternative cloning strategies There are many strategies beyond the...Addgene Blog 5 factors to help you choose the right cloning method Find blog posts about scientific sharing...

...posts on plasmid cloning Resources on Addgene.org Read our protocols for plasmid cloning Find our molecular... this scientists commonly use phagemids, a DNA cloning vector that contains both bacteriophage and plasmid...techniques in basic molecular biology, plasmid cloning, and viruses. References Baltrus, David A., and...

...incompatibility going to affect my cloning project?  If you want to clone two plasmids within the same bacterial...situation may arise for a microbiologist, where cloning two plasmids into one bacterium is not uncommon...incompatibility may be standing in your way to bacterial cloning glory, scientists have applied these concepts to...origin of replication Find blog posts about plasmid cloning Resources on Addgene.org Download the Plasmids...

...plasmids used in other systems, binary vectors are cloned and prepped in E. coli. However, they also need...large genes, smaller binary vectors with a larger cloning capacity would be very useful: enter pSIM24! To...customized due to its modular structure. Two multiple cloning sites (MCS's) flank the M24 promoter, allowing ...

...pBABE-puro (Plasmid #1764) Retroviral vector for cloning and expressing your gene of interest with puromycin...created pBabe writes how its name came about here. Cloning and expressing RNAi pMKO.1 puro (Plasmid #8452)... interest pLenti-puro (Plasmid #39481) You can clone your gene of interest into this tetracycline inducible...lentiviral expression vectors (empty backbones) here. Cloning and expressing new shRNA sequences pLKO (Plasmid...

... includes clone names, which can allow you to search different providers for the same clone. This can ...assessment of labeling pattern between multiple antibody clones targeting the same protein (independent antibodies...plasmids are available, or the necessary expertise to clone them, it can be useful to produce cell lines that...protein of interest. HLDA workshop approved antibody clones The antibody validation process represents a high...For CD365, they examined two different antibody clones produced by different vendors. The specific epitope...where different manufacturers may have the same clone available with different conjugates. Summary Flow...

...cell clones to use for multiple downstream experiments. You only successfully generate 1 null clone and...in that single clone is low, but since all downstream experiments will rely on that clone not having any... characterization of, isolating 2 or 3 distinct clones to perform key experiments on is a good way to ...

...Restriction Cloning Get Tips on Analyzing DNA Sequencing Results Check out Plasmid Cloning by PCR Resources...applications range from confirming the results of cloning or knockout experiments to forensics and clinical...Labs still keep restriction enzymes on hand for cloning and analytical digests. A researcher can test the...sequencing, such as confirming the results of a cloning experiment, require only one or a few sequencing...

...References Geertsma ER (2013) FX Cloning: A Versatile High-Throughput Cloning System for Characterization ...efficiently subcloned into the phagemid pDX_init plasmid, using the fragment exchange (FX) cloning technique...pools from the phage display screens are subcloned (using FX cloning) into the expression vector pSb-Init,...for identification. All unique sybodies can be subcloned into pBXNPH3 or pBXNPHM3 expression plasmids for...

...compared to other delivery methods. Retrovirus has a cloning capacity of ~8 kb, so either a single polycistronic...random integration creates variation between iPSC clones. Retroviruses are not considered safe for clinical...dividing and nondividing cells and have a larger cloning capacity (8-10 kb) than other retroviral vectors...cell, so there’s unavoidable heterogeneity between clones. This can complicate comparisons made between samples...between a vector and chromosomal DNA. PB has a cloning capacity of ~9-14 kb. This system is transfected...generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another challenge...for iPSCs Characterization Once generating iPSC clones, it’s important to determine how stem cell-like...

...Observing a bright signal for the first time after cloning an FP fusion with your favorite protein of interest...C-terminus [3]. You must therefore determine your cloning strategy and decide whether you will create an ... experimental needs. Equally important, before cloning you must consider whether and how well your FP ...peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila...