...Learn about the Cre-Lox system and how it can be used for genetic rearrangements and recombination. Use...

...BSL-2 Guidelines Remember, the BSL-1 laboratory guidelines above are expected to be followed in addition... PPE protocols . Working in a BSL-2 laboratory requires laboratory glasses in addition to coat and gloves... materials. Video Watch the video to learn more about safety in the lab. Equipment Lab coat Close-toed... eat, drink, chew gum, or apply makeup in the laboratory. Before you start your experiment, make sure ...Only mechanical pipetting should be done in the laboratory. Don’t mouth pipette! There may be some times...not be worn outside of the BSL-2 area. BSL-2 laboratories must be clearly marked as “BSL-2.” The names...names and contact information of the laboratory manager should be clearly visible in the room and on the ...

...Learn about selecting the correct pipettor and pipette tip, how to dispense liquids, and how to handle...liquid (think: 0.1 µL to 1 mL). When working in a laboratory, properly dispensing liquid ensures the accuracy...100 from top to bottom (as shown in the picture above), while 650 µL would read as 065. P200: The first...100 from top to bottom (as shown in the picture above), while 95 µL would read as 095. P20: The first ...10 µL would read as 100 (as shown in the picture above), while 2.2 µL would read as 022. P2: The first ...1 µL would read as 100 (as shown in the picture above), while 0.5 µL would read as 050. Each volume display...

...cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins. Optional...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr... of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample... of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration ...

...personal protective equipment including laboratory coat, laboratory goggles, and gloves. Sodium azide containing... not already been done. We typically start with about 250 mL of supernatant and add 250 µL of 100X protease... sample among multiple columns. For information about the total binding capacity for your columns, refer...setting. If the concentration of the pooled sample is above 1.0 mg/mL proceed to Option 1 with a buffer exchange...based on the concentration of the pooled sample above. Option 1: Buffer exchange using a desalting column...

... Protocols Video protocol guides for standard laboratory procedures. How-To Videos Screencapture walk-...Electrophoresis Gel electrophoresis is the standard laboratory procedure for separating DNA by size for visualization... MTA FAQs Answers to frequently asked questions about the MTA process Help Center How to Deposit Your ...projects in the works, and all of the things she loves about working at Addgene! Maria Soriano Maria Soriano,...in science, the importance of international collaboration, and offers career advice for scientists looking...

...and Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the Video...maintenance and use Watch the Video! Pipetting Learn about selecting the correct pipettor and pipette tip, ...the pipette Watch the Video! Centrifugation Learn about selecting and using a centrifuge to separate different... a liquid sample Using a Light Microscope Learn about the parts of a light microscope and its use Weighing...Weighing Reagents Learn how to weigh laboratory materials on a balance Basic Molecular Biology Name Description...

...Learn about selecting and using a centrifuge to separate different components in a liquid sample....Introduction A centrifuge is a common piece of laboratory equipment used to separate different components.... Floor Centrifuges sit on the floor and can be about bench height. They often spin at similar speeds ...tabletop microcentrifuge like the one in the picture above. Wear PPE appropriate for the lab space in which...

....8-fold. See the recipe for D1 + 0.1 M sorbitol above. Carefully pour off the media into a waste container...in a total of 5 mL of cell lysis buffer (recipe above). Pipet back and forth to resuspend each pellet ...pellets and keep on ice. Process the cell pellet from above as follows: Resuspend and lyse the cells by adding...tube containing the resuspended virus from step 12 above. Add 50 units of benzonase per mL of viral suspension...

...balance in the photo above has a capacity of 200 g, as indicated by the number above the "Zero" button. ... reagents for a stock mixture is an essential laboratory technique, as imprecise measurements can affect...

...water Microscope slide Anti-fade mounting medium Laboratory wipes 15 mL conical tubes 50 mL conical tubes...paraformaldehyde and follow your institution's laboratory safety guidelines for disposing of waste in the...remove the coverslip. Blot the coverslip with a laboratory wipe to remove excess liquid. Add 1 drop of anti-fade...

...Addgene’s Mammalian RNAi Tools page. Several other laboratories have deposited pLKO derived vectors that may...produce the same phenotype will alleviate concerns about off-target effects. B.2 Ordering Oligos Compatible...should be decontaminated with at least 10% bleach. Laboratory materials that come in contact with viral particles...work in a BL2 facility. If you have any questions about what safety practice to follow, please contact your...

...amplification process. When designing, if unsure about what nucleotide to put at a certain position within...capabilities. Taking into consideration the information above, primers should generally have the following properties...

...resistance and your initials. Labeling within a laboratory setting is important for organization, and it...a section of the plate, as shown in the diagram above, to create streak #1. Pro-Tips Hold your tooth pick...

...coli after Over-Agar Plating of Carbenicillin. The above graph displays the stock concentration of Carbenicillin...concentrations that will work for this assay, and the above result represents a single experiment. For publishable...

...Addgene lists the primers used to obtain each result above the posted sequence in the "View Sequence" link....sequence than expected and wish to contact Addgene about the accuracy of your plasmid, please email help@...

... lab! Introduction A water bath is a piece of laboratory equipment that helps bring your materials to ...even if you are using disinfectants as described above in step 3. You will also need to maintain the appropriate...

...verification, such as DNA sequencing . In the example above, digestion with enzyme RE1 will linearize the 6200bp...that you get double confirmation. In the example above, digestion with either RE3 or RE4 will give a very...

...research and are found in many different types of laboratories. These tools allow you to observe specimens ... in the future. Conclusion Like any important laboratory instrument, you should be sure to take care of...

...Information about plasmid cloning by restriction enzyme digest (subcloning), including design and experimental...window) New England Biolabs for more information about restriction enzyme buffers). If you select enzymes...

...approximately 50–60% confluent. For 293T cells this is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish ...cell solution into the conditioned medium prepared above to make a new cell solution at a concentration of...

... working with the volatile reagents in the list above. Procedure Option #1 - Solution D Protocol Before...sure to prepare solution D (see reagent section above for the recipe). If using TRIzol®, jump down to ...

...the titer. Calculations In the experimental setup above, the following virus dilutions were used to transduce...v = Virus volume, mL *In the experimental setup above, the cells per well is 300,000 and the virus volume...

...and bathroom. These items should remain in the laboratory. When traveling between lab spaces, take your...

...the now gel purified DNA. Find more information about DNA quantification here . Tips and FAQ How do you...

...water and quantify the concentration (should be about 8ng/μl). Mix the annealed oligos with cut vector...

...suggested amount, instead of the other dry ingredients above. Media without growth (top) and with growth (bottom...

...more at (Link opens in a new window) NEB's website about star activity . If you are digesting a large number...

... the right). When designing your plasmid, think about what DNA segments you will need to join to create...

... supernatant out of the tube if you are worried about losing the pellet. Dry with vacuum or by inverting...

...manufacturer’s instructions for specific instructions about extension time and temperatures. Initial Denaturation...

...cells in the untransduced well (0 µL lentivirus, above) are dying. Perform regular media changes and monitor...

...Background In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same ...

...point. If your unknown sample’s absorbance falls above the range of the standard curve you will need to...

...dilution BSC. Prepare 1X dilution buffer (see recipe above). Save ~25 µL of 1X dilution buffer in a microcentrifuge...

...require modifications dictated by the originating laboratory for optimal results. If you obtained the pooled...

... | Fourth Edition About Plasmids 101 September 2023 | Fourth Edition About Based on our popular...techniques! Close About Overlay Select CRISPR 101 January 2021 | Third Edition About CRISPR 101 January...screens. Close About Overlay Select Fluorescent Proteins 101 August 2017 | First Edition About Fluorescent... more! Close About Overlay Select Viral Vectors 101 July 2024 | Second Edition About Viral Vectors...more! Close About Overlay Select Science Career Guide November 2018 | First Edition About Science Career...Close About Overlay Select CRISPR 101 en español January 2024 | First Edition, trans. About CRISPR... trans. About Una traducción al español de nuestro libro electrónico, CRISPR 101. Close About Overlay ...

...are often used in clinical applications. Diabodies - Diabodies contain two Fab fragments, each recognizing...cells isolated from animals immunized as described above are used to create hybridomas, which produce large...different epitope, bound by short peptide linkers. Diabodies can be used for assembling protein nanostructures... Includes: Flow cytometry Read on to learn more about the applications common to each category, including...controls, and any special considerations to think about. For step-by-step instructions of many of these ...protein of interest, as described in the IP section above. Reverse the crosslink, isolate DNA, and perform...basic protocols and controls follow that of IF (above). Sample preparation, signaling excitation, and ...

.... However, CNO is back-metabolized to clozapine and other clozapine metabolites and these can have off-target...concentrations of clozapine N ‐oxide (CNO). CNO is a metabolite of the antipsychotic clozapine and seems to be...experiments in this species may be entirely due to back-metabolism of CNO to clozapine, demonstrating the need for...activity, and do not seem to have the same back-metabolism issues as CNO and are attractive alternatives...chemical variations thereof (FFz, CFz). Learn more about optogenetic systems in our Optogenetics Guide . ...tissue-specific Cre is provided. Want to know more about FLEx vectors? Read our Plasmids 101: FLEx Vectors...

...Production Schematic Frequently Asked Questions (FAQ) about Lentiviral Plasmids What is the difference between...lentiviruses, please review 2nd vs 3rd generation above . Briefly, 2nd generation lentiviral systems use... useful restriction sites. For more information about cloning and changing a MCS, visit Addgene's Plasmid...addressed by the design of the vectors and by safe laboratory practice. In terms of vector design, 2nd and ...round of reverse transcription. This deletion abolishes transcription of the full-length virus after it... JM, Hughes SH, Varmus HE. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, 1997. Available online...

...Where can I find information about using the AdEasy™ System? The laboratory of Bert Vogelstein has published...linearized construct, and virus can be harvested about 7-10 days later. Vogelstein designed multiple shuttle... Return to Top Frequently Asked Questions (FAQ) about Adenoviral Vectors Are adenovirus and AAV different...E2a and VA for replication. For more information about AAV, read our AAV guide . Can I make a stable cell...

...human genome with restriction enzymes would yield about two million DNA fragments, which is far too many...the molecular biologist’s toolbox. To learn more about different types of cloning methods check out our...restriction enzyme digestion. For more information about restriction enzymes check out NEB's website . Insert...endogenous expression levels). For more information about promoters, both bacterial and eukaryotic, as well...can be replicated easily and efficiently in a laboratory setting. Stable - Plasmids are stable long-term...codon. Below you will find helpful resource tables about the genetic code. This table includes the nucleotide...

...biology, laboratory research, and career development. eBooks Download any of our eBooks to learn about popular...Playlists include: Protocols Careers How-to Blog Read about plasmids in Addgene’s repository , and related molecular...Technology Career Readiness Molecular Biology Learn about plasmids, sequencing, vectors, and restriction enzymes...

...Learn about the types of plasmids used to produce AAV in the lab. Addgene’s AAV Guide will teach you ...you about AAV components, serotypes, pseudotyping, and more....non-homologous sites in the genome, and do so at about a 0.1% frequency. Nonetheless, the majority of rAAV...enhanced brain uptake. AAV Variants As mentioned above, AAV improvements have included the production of...

...packaging methods. The three components described above (envelope, packaging, and transfer) are supplied... Return to Top Frequently Asked Questions (FAQ) about γ-Retroviral Plasmids How do engineered γ-retroviruses...may be used interchangeably. For more information about lentivirus, please read our lentivirus guide. Are...

...and are discussed in more detail later. Read more about Cas9 multiplexing . Browse Plasmids: Multiplex gRNA...Nme2Cas9 (from Neisseria meningitidis ), which are about the same size as SaCas9, with higher fidelity than...targeting range for plant genome engineering. Read more about Cas9 variants . Anti-CRISPR Prolonged Cas9 activity...convert one base to another within a target locus of about 8 bp, specified by a gRNA. There are two main types...sequence may not be characterized *In the table above, 3' and 5' indicate on which end of targeted sequence...

...components to make lentiviral particles (for details about lentivirus, see our Lentivirus Guide ) Stable, tunable...when carrying out CRISPR experiments. Read more about how to design your gRNA . Browse Plasmids: gRNAs...to optimize your delivery conditions. Learn more about CRISPR delivery in mammalian systems . Validate ...

...the following guides to help you start learning about various scientific concepts related to our plasmid...Viral Vectors Find educational guides to learn more about the different types of viruses that are commonly...

... to conduct a pooled library screen. The figure above is an outline of how a gRNA library is used. Below...simultaneously in a single reaction. As discussed above, access to this technology is crucial for conducting...

...of interest is expressed. Summary Promoters are about 100-1000 base pairs long and are adjacent and typically...Inducible by arabinose Promoter of the arabinose metabolic operon trp Repressible by tryptophan Promoter ...