...plasmid. Designing Primers for PCR Based Cloning The basic PCR primers for molecular cloning consist of... Protocols Plasmid Cloning by PCR Plasmid Cloning by PCR You may also...PCR based cloning carries a much higher risk for mutation than restriction enzyme based cloning. DNA replication...Protocols for plasmid cloning by PCR. Protocols...Ligation Bacterial Transformation Summary PCR based cloning is incredibly versatile and allows for nearly any...limitations. Background In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at...Restriction Site: Your chosen restriction site for cloning (usually 6-8bp). Hybridization Sequence: The region...

....1-TRC Cloning Vector A.1 The RNAi Consortium A.2 Map of pLKO.1 A.3 Related plasmids B. Designing shRNA...Digesting pLKO.1 TRC-Cloning Vector C.4 Ligating and transforming into bacteria D. Screening for Inserts D.1...pLKO.1 TRC-Cloning Vector I.2 Recipes I.3 Warranty information Back to Top A. pLKO.1-TRC Cloning Vector A...Figure 1 : Map of pLKO.1 containing an shRNA insert. The original pLKO.1-TRC cloning vector has a 1.9kb stuffer...vector containing non-hairpin insert. pLKO.1 – scramble shRNA Negative control vector containing scrambled...for “pLKO”“. Back to Top B. Designing shRNA Oligos for pLKO.1 B.1 Determining the Optimal 21-mer Targets...these bases are important for cloning the oligos into the pLKO.1 TRC-cloning vector. Forward oligo: 5’ CCGG...

...Changing a Multiple Cloning Site (MCS) Adding short tags - ex: HA or Flag Cloning shRNAs Background For... MfeI. With oligo overlap cloning, you can design a set of oligos containing your desired restriction ...using oligo overlap cloning. Protocols... Protocols Plasmid Modification by Annealed Oligo Cloning Plasmid Modification...Modification by Annealed Oligo Cloning You may also like... Restriction Digest of Plasmid DNA DNA Ligation Bacterial...Bacterial Transformation Summary Oligo overlap cloning can be used anytime you need to add a short stretch...digest of existing sites in the original vector. Designing oligos To add NdeI, PacI, AscI and MfeI sites ...

... Protocols Subcloning Plasmid Cloning by Restriction Enzyme Digest (aka Subcloning) You may also like...Information about plasmid cloning by restriction enzyme digest (subcloning), including design and experimental...out our Empty Backbone Reference . Background Subcloning by restriction digest is a commonly used lab ...element between plasmids. Let's assume that you are beginning a new project on your gene of interest (YGOI for...YGOI is in a bacterial expression vector. Using subcloning, you can easily move YGOI into a mammalian expression...your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid...find two different restriction enzymes for your subcloning. It is also possible to use a single enzyme, ...

...Plasmid Cloning Name Description (Link opens in a new window) Link to Video Restriction Cloning Assemble...ligation Cloning by PCR Generate restriction sites by PCR Modification by Annealed Oligo Cloning Add a short...single reaction Ligation Independent Cloning (LIC) Scarless cloning with Type II restriction enzymes and...and T4 polymerase pLKO.1 - TRC Cloning Vector Cloning protocols for using the pLKO.1 vector, a backbone...can be used for basic molecular biology, plasmid cloning, and titering and testing your viral preparations... for many more complicated procedures. Plasmid Cloning Protocols for constructing and analyzing your plasmids...How to Design a Primer Key considerations when designing primers Watch the Video! Sequence Analysis Verify...

...pAAV-CAG-iSeroSnFR-Nlgn Fluorescent reporter for serotonin (dendrite localization tag) Lin Tian 135420-AAV1...

...purpose: fast growth, high-throughput cloning, routine cloning, cloning unstable DNA, preparing unmethylated...Streptomycin MC1061 derivative. General cloning and storage, blue/white screening, leucine auxotroph. F- endA1 recA1... DH5alpha   General cloning and storage of common plasmids, blue/white screening. F- endA1 glnV44 thi...glnV44 λ- JM109   General cloning and plasmid maintenance, blue/white screening, partly restriction-deficient...NEB Stable   For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...JM109-derived. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...from HB101. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...

...Direct staining is quicker than indirect staining and eliminates potential non-specific staining that may...can use both direct and indirect staining methods. In direct staining, the primary antibody against a ...intracellular staining where binding of large immune complexes may be hindered. Indirect staining can be beneficial...also keep their targets in mind when planning fixation. If staining both cell surface and intracellular...control is critical for determining the background level of non-specific staining.  If you’re using antibodies...-specific staining associated with the primary antibody. When following an indirect staining protocol,...omitted from the staining protocol. This control allows you to detect background staining arising from the...

...students, rotation students, joining a new lab at any career stage, learning a new technique while visiting...participant in the training. In this blog we will review the preparation and planning you can do to build...true for training in the lab! If you don’t do proper preparation and pay attention during training, then ...questions to your training! All of these things will help you get more out of the in-person training component...need to double-check a detail! Training follow up After your training session (ideally in the next few... the information in your training. Bench mentor-mentee relationships Training can be a very fulfilling...visiting a lab, or maybe even training on a new technique with a coworker. Being a new trainee (even if you...

...constructs? A new cloning system combining the advantages of Golden Gate and Multisite Gateway cloning methods ...that you need. The Golden GATEway cloning kit simplifies the cloning process for complex DNA constructs...vectors via traditional restriction enzyme cloning, TA cloning, or annealing of oligonucleotides. These ...efficient and flexible cloning process provides improvements to classical cloning methods, particularly... MoClo modular cloning system Synthetic biologists have developed a modular cloning strategy, MoClo,...assembly steps. Before beginning, scientists can insert fragments of DNA containing basic parts (promoters... of interest are retained after SLIC cloning. The general cloning strategy described in the associated...

...building block for further chaining. The MXS-chaining kit contains 5 empty chaining vectors plus a destination... 19; 22(2):169-74. High efficiency cloning for enzyme screening Dr. David Ackerley’s lab at the University...expression containing a variety of N- and C- terminal tags. The Santalucía Lab has developed a cloning toolkit...Multisite Gateway cloning. A desired insert is cloned using traditional restriction enzyme cloning to create ...(5):493-8. MXS cloning platform for imaging and flow cytometry applications Designing customized fluorescent...Neveu laboratory designed a new iterative, chaining-based cloning method that simplifies the process of constructing...with MXS-chaining. Analogous to modular assembly methods, such as BioBricks, the MXS-chaining method uses...

...any well-executed cloning endeavor, NEBuilder requires upfront planning. This cloning methodology requires...? In the context of cloning, DNA assembly refers to a method of physically joining multiple fragments ... the joining reaction. BioBrick Assembly will leave scar regions at the site of fragment joining, but ... for seamless joining of multiple mutations spread over thousands of base pairs. Cloning sgRNAs Generating... More about Gateway Cloning Gibson Assembly and Other Long-Homology Based Cloning Methods Screen Plasmids...end of a fragment. This means that fragments (containing regions of homology) cut from plasmids via restriction...the regions of homology. A powerful resource for planning an NEBuilder HiFi DNA Assembly mix reaction is...

...process of cloning, combining, and testing these different parts can be quite arduous. The cloning process...of a good screening experiment can be found on our blog post entitled “Genome-wide Screening Using CRISPR...Moduclar Cloning (CIDAR MoClo) Parts Kit. This Kit consists of a variety of plasmids containing many different...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...GPCRs in the human genome are orphan receptors meaning their ligands are unknown. Identifying new ligands...approach termed Parallel Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO...

....  The partitioning problem Plasmid incompatibility also occurs because of plasmid partitioning (Schumacher...within one cell have the same replicon and/or partitioning system. Let’s start with the replicon- the part...to initiation replication. However, in iteron-containing plasmids,  RepA can also bind to its own promoter...a concern with low copy plasmids, though the partitioning problem does occur in high copy plasmids (Diaz...are several different versions of the plasmid partitioning system in the bacteria, but generally it consists...For high copy plasmids, incompatibility due to partitioning is similar to incompatibility due to having ...loss (Diaz et al., 2015).  Figure 2: The partitioning problem. For low copy plasmids, the main ...

...Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally...then you might consider designing orientation-specific primers. Blunt end cloning is an example of when ... Addgene Blog Learn to do Restriction Cloning Try SLIC cloning Learn about Gibson Assembly Additional...things to consider when performing colony PCR.   Designing colony PCR primers The first and perhaps most ...most important step to colony PCR is designing primers. There are 3 strategies for primer design: 1) insert-specific...backbone. This type of primer pair is also great for screening clones created with the same backbone but that... When you design primers to anneal outside the cloning site, it doesn’t matter what the sequence of the...

...for an example image). Point Scanning Confocal Microscopy Point Scanning Confocal Microscopy was the first...microscopy technique to incorporate optical sectioning. Optical sectioning refers to the ability to extract light... Confocal Microscopy (Spinning Disk) Parallelized Confocal Microscopy (Spinning Disk) increases the speed...to enhance performance. In spinning disk microscopy, a metal disk containing a number of holes rotates ...samples always require some form of optical sectioning. Point scanning confocal microscopes (see above) often...volumes to enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...excitation light to a point in the sample and raster scanning the point to build up a final image pixel by pixel...

...transitions are edits between the purine bases (adenine, guanine, and hypoxanthine) or between the pyrimidine...glycosylase (MPG), also known as alkyladenine DNA glycosylase (AAG). When adenine base editors (ABEs) were originally...BioRender.com. Inosine excision leads to the first adenine transversion editors Uracil excision is the most...endogenous enzyme is not efficient enough to hinder adenine base editing. Once base transversion editors were... able to edit A → Y (Y = T or C) in 67.17% of screening cells (Tong, Wang, et al., 2023). Based on this... team used rational mutagenesis to produce the adenine transversion editor AYBEv3. This tool still retained...A → C product purity of 48%. Like CGBEs, these adenine transversion editors produce a lot of indels (up...

... analytic scientists seem to toss data-driven reasoning out the window when making decisions. We think...decisions without much logic - even choosing a training lab where we will spend the next 6 years. Know...very communal. That didn’t suit my collaborative learning style at all. Finally I rotated in a small lab...about this lab’s reputation? If you are choosing a training lab, I strongly suggest you complete a work rotation... the same field taking projects with them and remaining close colleagues of the PI? Or do lab alumni leave...they “needed it more”. My friend didn’t heed this warning and profoundly regretted it when the same thing...year later. I hear too many science trainees get warnings and still think “but that won’t happen to me, ...

...of scientist training in the US and around the world. There are rumors that we are training too many scientists...opportunities after training. One of the best recent discussions of this apparently worsening crisis recently...the way we view the graduate school and postdoc training years. There is no doubt that there are too many...academia. Certainly funding for academic research and training is getting tighter and competition is fierce. ...sufficiently prepared during their 6-12+ (!) years of training to get jobs. They are not exposed to the vast ...radical (and some not so radical) suggestions for defining the problem and for finding solutions. Even better...meeting new people by working together on the event planning and execution. I am seeing many similar examples...

...reporter. The only caveat is that HA staining is weaker than reporter staining. Picking the right reporter for... them. HRP RANbody staining results in a ≥10-fold increase in signal over staining with a primary/ labeled... sensitive than HRP-RANbody staining Requires secondary antibody staining HA Tagged GFP, RFP, H2A2B ... IgY antibody Staining cultured cells and tissue sections Great for multicolor staining of tissue since... sensitive than HRP-RANbody staining Requires secondary antibody staining GFP, RFP, H2A2B   Are you...smaller size of RANbodies compared to antibodies. Staining with multiple HRP-RANbodies is indistinguishable... domains are especially useful for multi-color staining of tissue because they have limited cross-reactivity...