... (AAV) Guide Components Common Uses Viral Integration Serotypes Pseudotyping AAV Variants References AAV... enhanced brain uptake. AAV Variants As mentioned above, AAV improvements have included the production... Addgene’s AAV Guide will teach you about AAV components, serotypes, pseudotyping, and more. Science... AAV Components The small (4.8 kb) ssDNA AAV genome consists of two open reading frames, Rep and Cap, ... of AAV AAV is commonly used in optogenetics experiments. These viruses are preferred over lentiviruses...has been modified for use with AAV; this system represents a major step forward for in vivo genome editing...hybrid viruses with new properties. Other system variants include: Self-complementary AAV (scAAV): One downside...

... methods. Across these methods, the followimg components are needed: γ-Retroviral transfer plasmid encoding...similar to lentiviral packaging methods. The three components described above (envelope, packaging, and transfer...slightly different isoforms of these packaging components. Therefore, lentiviruses may not be efficiently...plasmid backbone is meant to be descriptive of the components found within the backbone. For example: pLXSN...particle containing Matrix, Capsid, and Nucleocapsid components. Pol in trans Precursor protein containing Reverse...Reverse Transcriptase and Integrase components. Transfer cPPT in cis Central polypurine tract; recognition...transgenes via increased nuclear export. LTR Subcomponents: U3 R U5 in cis LTR; Long terminal repeats; ...

... RNA polymerase. DNA sequences called response elements are located within promoter regions, and they ...regulatory sequences including enhancers, boundary elements, insulators, and silencers. Promoter Regions There... package DNA into nucleosomes. Histone binding prevents the initiation of transcription whereas transcription...promoter which contains many primary regulatory elements. The proximal promoter is found approximately ... binding sites, but mostly contains regulatory elements. Eukaryotic Promoters Eukaryotic Transcription.... It is not unusual to have several regulatory elements such as enhancers several kilobases away from ...are normally bound by a repressor protein that prevents transcription. When an inducer molecule binds ...

...adenoviral components and vector construction... Science Guides Adenoviral Guide Adenoviral Guide Components Vector...Frequently Asked Questions (FAQ) Glossary Adenoviral Components The adenovirus genome is a linear, 36-Kb double-stranded...essential for virus production. Deletion of these two components results in a transgene packaging capacity of ...911 cells throughout virus production. During experiments, GFP can be used to sort cells infected with ...propagate your virus as the chance of recombination events creating replication-competent virus increases ...Definition ITR Inverted terminal repeat; Contains elements that promote bi-directional transcription Ad5-...

...you to design additional hypothesis-directed experiments. As you might have guessed from their name, pooled... false positives or false negatives in later experiments, and can also negatively affect data reproducibility...only way to accurately evaluate which library components are influenced by selection, so it’s essential...allows for parallel sequencing of millions of DNA fragments simultaneously in a single reaction. As discussed...

...barrier in rats, and the utility of DREADDs in experiments in this species may be entirely due to back-metabolism...issues as CNO and are attractive alternatives for experiments. Perlapine has been previously used in human ...respond to the same ligands was that bidirectional experiments (e.g. using both activating and inhibiting DREADDs...absence of Cre and are especially useful in AAV experiments where the virus can spread around the injection...

...applications, tips for using vectors in your experiments, troubleshooting advice on production and use...

...Join almost any two fragments regardless of sequence. No scar between joined fragments. Fewer steps. One...about what DNA segments you will need to join to create your final plasmid. Adjacent segments should have...If there are significant amounts of undesired product, gel-purify DNA segments. Otherwise, PCR purification...combine segments in the Gibson Assembly Reaction. Pro-Tip Yields will be best when the DNA fragments are ...annealed DNA fragments. Taq DNA Ligase - covalently joins the annealed complementary DNA fragments, removing... al. , 2010). Number of fragments assembled simultaneously Multiple fragments can be assembled in one ... for the easy assembly of multiple linear DNA fragments (Gibson et al. , 2009). Regardless of fragment...

...Matthew Nolan 138134 pT7NT*-Bri2 113-231 R221E ITM2B NT T7 Familial British dementia Jan Johansson 138347...

...bottom. For the P1000, this represents the ones. For the P200, this represents the first decimal place. ...For the P20, this represents the second decimal place, and for the P2 this represents the third decimal...in many scenarios to accurately dispense small amounts of liquid (think: 0.1 µL to 1 mL). When working...microfuge tube, bottle, etc.) Labels for containers Reagents Liquid for pipetting Background Information Anatomy... container in order to avoid contaminating the contents. Once the end of the tip is submerged, slowly ...small changes in the volume could affect your experiments. Beyond knowing the proper way to pipette, it...they are properly tuned to dispense the correct amounts of liquid. If you are pipetting the same amount...

...selection in your experiments. (You may wish to repeat this titration with finer increments of puromycin to...See “warranty information” in appendix. Table of Contents A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium...microcentrifuge tube. TIP: When visualizing DNA fragments to be used for ligation, use only long-wavelength...products on a 1% agarose gel. You should see two fragments, a 2kb fragment and a 5kb fragment. 5. Sequence...the host genome. For stable loss-of-function experiments, Addgene recommends that you generate lentiviral...The protocol can be scaled to produce different amounts of virus as needed. Day 1: a. For each plasmid ...puromycin should be from 1-10 μg/mL in 1 μg/mL increments. d. Label plates from 1-10 and add appropriate...

... that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands...matrix toward a positive electrode. Shorter DNA fragments migrate through the gel more quickly than longer...gel alongside a DNA ladder (a collection of DNA fragments of known lengths). Last Update: Feb. 20, 2018 ...Voltage source Gel box UV light source Microwave Reagents TAE ( recipe here ) Agarose Ethidum bromide (stock...Another method for visualizing very short DNA fragments is polyacrylamide gel electrophoresis (PAGE), ...which is typically used to separate 5 - 500 bp fragments. How do you get better separation of bands? If...

...specifically uses primers and probes targeting the ITR elements in the viral vectors but can be modified for other...GC/mL, where GC is genome copies and generally represents the physical number of viral particles. Users...Before Starting To reduce the risk of contaminating reagents we recommend making small aliquots of master mixes...10199-452 Ice bucket 96-well freezer blocks (x 3) Reagents Molecular Biology Grade Water, Hyclone, SH30538.02...generation BSC) to ensure no contamination of the PCR reagents and the NTC with virus. Pre-warm the 96-well plate...equipment with 10% bleach prior to use and keeping all reagents and samples on ice or pre-chilled 96-well freezer...accuracy of the titer, take the average of several measurements. Figure 1: ddPCR sample data Sample Dilution...

...analyze the resulting fragments by gel electrophoresis . The pattern of the fragments on the gel can indicate... tips Reagents Liquid DNA aliquot of your plasmid of interest (see below for recommended amounts) Appropriate...plasmid into 3-8 pieces such that all (or most) fragments are small enough to be accurately sized on a gel...enzymes that will cut your plasmid into multiple fragments, you can get a very unique pattern that will distinguish...

...rearrangement of the plasmid DNA. Additionally, specific treatments have been discovered that increase the transformation...important if you will be transforming with very small amounts of DNA or if you're multiple plasmids at once. ...Microcentrifuge tubes Sterile spreading device Reagents LB agar plate (with appropriate antibiotic) LB...colonies, while allowing you to recover all transformants. If the culture volume is too big, gently collect... at least 20-30 mins) I get very few if any transformants when transforming large plasmids (>10 kb) or...manufacturer's instructions for each. I got no transformants. What went wrong? Check that you are plating...

...electron microscopes that allow us to see cellular components with science-fiction levels of detail. You can... of a compound light microscope with the main components labeled: Figure 1: Diagram of a compound light... make it through the sample because different components of the specimen will refract and reflect the ...) using dyes or stains that add color to some components of the sample but not others. The light that ...other sample that can fit on a microscope stage) Reagents None needed Procedure Set up your microscope by...the smaller of the focus knobs) to make minor adjustments to the focus. After you are happy with the positioning...

...invert the tube carefully 5 times to mix the contents. The contents will become clear and thicker as the proteins...microcentrifuge Desktop vortexer Vacuum (optional) Reagents Overnight culture of bacteria transformed with...step causes the bacteria to lyse, releasing their contents, including plasmid DNA, into solution. Add a renaturing...,000 g. Notes: Pellet contains proteins, cell fragments, salt and other extra particles from solutions... This will help to remove proteins and other contaminants from the plasmid DNA. It is also advisable to...

...barcoding, or other high throughput multiplexed experiments. These pooled plasmids differ by only a short...resulting in a smaller plasmid containing only the elements required for bacterial propagation (origin of ...Electroporator (MicroPulser TM , Bio-Rad 1652100) Reagents 200 µL electrocompetent cells (Default: 4 tubes...of cells. Ensure access to autoclaved, sterile reagents for all steps. Procedure Day 1 Add 200 ng DNA ...cells, more DNA, gentler practices, and colder reagents (not including the recovery media!). Do not proceed...and sometimes fall off entirely if the column or reagents are significantly overloaded. If you find that...

... connecting the sugar backbone of the two DNA fragments. This reaction, called ligation, is performed ...The majority of ligation reactions involve DNA fragments that have been generated by restriction enzyme...insert will be added in the correct orientation and prevents the vector from ligating to itself during the ...phosphatase removes the 5' phosphate and therefore prevents the ligase from being able to fuse the two ends...of intact plasmid in ligation or transformation reagents Optimizing the Vector:Insert Ratio Although a ...

...protocol is composed of steps that separate out contaminants from an impure AAV preparation. The 15% iodixanol...macromolecules. The 40% and 25% steps are used to remove contaminants with lower densities, including empty capsids...Equipment Ultracentrifuge T70i rotor or equivalent Reagents Opti-Prep (60% Iodixanol) QuickSeal tubes QuickSeal...have a good idea when the virus elutes from the gradients. In a biosafety cabinet, carefully puncture the...by silver stain. Note the increased number of contaminants in each fraction. * AAV capsid proteins VP1,...