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We narrowed to 363 results for: nsf

Showing: 41 - 60 of 363 results

  1. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    Published
    Oct. 23, 2018, 11:49 a.m.
    ...XmaI restriction sites). Another way is to transform AAV transfer plasmids into bacterial strains, like NEB...Overview: The first step in packaging AAV is transfecting HEK293 cells with AAV packaging plasmids. The...help mediate AAV viral replication; and 3) the transfer plasmid which encodes a transgene of interest ...packaged into the virus. Two to five days after transfection, the AAV-containing cells and media are harvested...more details. Pro tips I’m not going to lie, AAV transfer plasmids are kind of a pain to work with. These...structures that are prone to deletion from the transfer plasmid. The good news is there’s a few simple...to grow multiple bacterial cultures of your AAV transfer plasmid at 30 °C instead of 37 °C and then screen...

  2. Viral Vectors 101: Transductions

    Type
    Blog Post
    Published
    April 30, 2024, 1:15 p.m.
    ...The triple T’s of biology are transformations, transfections, and transductions! In this blog we will...lipid-based or electrical-based transfection, most of the nucleic acid transfected will not stably integrate...picky when it comes to transfection/transduction methods. Some ‘difficult to transfect’ cells lines are amenable...transduction?  Transduction is the process of transferring foreign nucleic acid into a cell by a viral ...viral agent. Transductions are similar to transfections, processes which introduce foreign nucleic acid into...they require delivery to be viral-mediated. Transformations are usually bacterial in nature. All three ...containing the cargo of interest is then typically transfected along with viral packaging plasmids into a viral...

  3. The MTA at Addgene

    Type
    Blog Post
    Published
    July 23, 2024, 2:17 p.m.
    ...Biological Material Transfer Agreement (UBMTA) is one of the most commonly used ones for transfers between academic...for more information. Happy transferring!    Many thanks to our Tech Transfer team members Maura Coughlin...Addgene, then you’ve been involved in the material transfer agreement (MTA) process. Even though this process...curious about the hows and whys of the material transfer agreement, read on!  What is the MTA?  An MTA...the creation of a standardized agreement on the transfer of biological materials in 1995: the UBMTA. Because...most academic and nonprofit institutes, the Tech Transfer department is responsible for managing research...plasmid with Addgene, your organization’s tech transfer office is sent a deposit agreement. The One-Time...

  4. Plasmids 101: The Wide World of Natural Plasmids

    Type
    Blog Post
    Published
    Oct. 8, 2024, 1:15 p.m.
    ...lateral”) gene transfer. Horizontal gene transfer can occur naturally through transformation (cellular uptake...? Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection Resources on Addgene.org...infection by bacteriophages), or conjugation (direct transfer from one cell to another). Bacteria need special...one popular method scientists use to promote transformation, but it isn’t just for lab specimens — there... evidence that lightning can also promote gene transfer in soil bacteria! (Demanèche et al., 2001) Beneficial...humans only noticed last century! Nevertheless, the transfer of plasmids carrying these genes between bacteria...plasmid are now used routinely in biotechnology to transform plant cells. Degradative plasmids: microbial hardiness...

  5. Lab Automation at Addgene

    Type
    Blog Post
    Published
    March 22, 2018, 1:28 p.m.
    ...bacterial transformation One protocol that has been automated is high throughput transformation. With our... to do up to 96 transformations in one run compared to doing about 50 transformations in the same amount...easier and more efficient. Here, I’ll discuss transformation, DNA purification, and filling vials with agar...deposits (over 100 deposits per month!), manual transformations were taking a lot of time. Enter the Biomek... We wanted a machine that would improve our transformation protocol and, after looking at a few options... - one pod can pick up 96 tips for easy sample transfer between 96 well plates. The second pod can pick...out your samples. This can be useful if you’re transferring samples from 96 wells plates onto agar q-trays...

  6. CRISPR 101: Mammalian Expression Systems and Delivery Methods

    Type
    Blog Post
    Published
    Sept. 24, 2015, 2:30 p.m.
    ...be present in a single lentiviral transfer vector or separate transfer vectors. May contain reporter gene...Chemical transfection   Lipid mediated Cationic polymers Calcium phosphate Cells Transformed cell lines... lines Transformed cell lines Transformed cell lines Cargo Nucleic acids or protein Nucleic acids Nucleic...system. CRISPR reagents can be delivered via transfection, nucleofection, viral transduction or injection...or gRNA in a mammalian cell line that can be transfected at high efficiency. Lentiviral transduction...mammalian cell lines. Useful for difficult to transfect cell types and can be used in vivo. A common choice...4.5kb). CRISPR elements are inserted into an AAV transfer vector and used to generate AAV particles. Transient...

  7. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    Published
    May 27, 2025, 1:15 p.m.
    ...BioRender.com. Transformation Grade DNA (low quality/low quantity) – Bacterial transformation protocols require...highest-competency cells you will still struggle to get transformants if the plasmid you are using is completely degraded...important reactions like PCR or restriction digests. Transfection Grade DNA (high quality/high quantity) – Depending...Depending on the experiment, mammalian cell transfections can require quite large amounts of DNA (easily...that may not be explicitly labeled as “yeast”, “transfection-grade”, or “large plasmid” may be suitable for...automation-friendly Not mentioned Endotoxin-free, transfection-grade, BAC Millipore Sigma Column, plate Mini...automation-friendly Mentioned Endotoxin-free, transfection-grade Promega Beads, column, plate Mini, midi...

  8. Fluorescent Proteins 101: Introduction to FRET

    Type
    Blog Post
    Published
    June 27, 2017, 1:03 p.m.
    ...this FRET you speak of? Förster Resonance Energy Transfer (FRET) was originally described by Theodor Förster...alternative acronym Fluorescence Resonance Energy Transfer. Unlike the typical excitation and emission of...excited fluorophore, FRET involves a non-radiative transfer of energy (i.e. no emitted photons) from the excited...excitation by absorption of a photon 2. Energy transfer from the excited donor directly to the acceptor...the acceptor fluorophore. Otherwise the energy transferred from the donor will not be able to excite the...other and be oriented appropriately for energy transfer via dipole-dipole interaction. The efficiency ...al. "Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant...

  9. FastCloning: A Newer, Simpler Cloning Technique

    Type
    Blog Post
    Published
    Nov. 1, 2016, 2:30 p.m.
    ...products are then transformed directly into chemically competent E. coli and transformants selected with ... for further purification before transformation. After transforming cells, you should perform colony ...consisting of digestion, purification, ligation, and transformation. While restriction cloning is still routinely...+ + Gel Purification + - Ligation + - Transformation + + The FastCloning process in brief In ...for this procedure to work properly. Directly transform chemically competent cells with the digested mixture...-cycle PCR leads to the most colonies after transformation. When a 1:1 vector:insert ratio is used in ... and 2 µL of digest product are used in the transformation, >70% of the colonies are correct. Good luck...

  10. Viral Vectors 101: Gamma-Retroviral Packaging Systems

    Type
    Blog Post
    Published
    Aug. 6, 2024, 1:15 p.m.
    ...packaging, and transfer) are supplied by three types of plasmids, which are cotransfected into the 293T...following components are needed: γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA ...sequences, which facilitate integration of the transfer plasmid sequences into the host genome. Typically... the host genome upon viral transduction. Most transfer plasmids available from Addgene were derived from... number of plasmids that are required in the transfection step of the viral packaging cell line. For example...packaging and transduction are not encoded by the transfer plasmid, but instead are provided in trans by ...Reverse Transcriptase and Integrase components. Transfer cPPT Central polypurine tract; recognition...

  11. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    Published
    April 17, 2018, 1:37 p.m.
    ...The typical drawbacks of using transfection will still exist: transfection efficiency is cell-type dependent...cells Limited genomic integration Multiple transfections required Need to sequence to verify there’s ...High Transgene-free and vector-free Multiple transfections required Protein Delivery DNA-Free Low High...cloning capacity of ~9-14 kb. This system is transfected into cells as a donor plasmid containing the ...with plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming...describes a protocol for generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration...integration. Another challenge is that multiple transfection makes it difficult to control the dose of plasmid...

  12. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    Published
    March 13, 2014, 4:08 p.m.
    ...positively transfected. Alternatively, you can use a selection marker to select transfected cells (for..., the most efficient and easiest way is to co-transfect several plasmid together, with each plasmid having...two spacers for each locus into PX330, then co-transfect or delivery the two plasmids together into your...run them, and then select the best guide to co-transfect in HR donor. Alternatively, a new system described...oligo is fine based on new data). For general transfection, you could use 1 µl of 10 µM oligo stock for... or mutation. Each arm is around 800 bp. For transfection in cells like HEK, we use around 400 ng of a...single-point mutation, the best HR template for transfection is probably a single stranded DNA (ssDNA) oligo...

  13. 5 Tips for Troubleshooting Viral Transductions

    Type
    Blog Post
    Published
    Aug. 11, 2016, 2:23 p.m.
    ...immediately following transfection. In other words, when exchanging the medium after transfecting cells, replace...can check if virus is being generated by your transfected packaging cells. This check can be performed ... your packaging cells have been successfully transfected and produced infectious virus, they may get infected...can be performed approximately 72 hours after transfection and should give rise to a surviving population...plasmid integration via recombination during transfection, and does not necessarily indicate the production...protein or resistance marker expression following transfection, higher-level expression after approximately... Yarmush. "Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus...

  14. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    Published
    June 28, 2018, 3:01 p.m.
    ...complex. This complete transfection mix, i.e. csRNP plus ssODN, is then transfected into stem cells. Cells... were transfected in 96-well format using an Amaxa Shuttle device; 5 days after the transfection, the ...Prepare cells for transfection, add csRNP and ssODN to the cell suspension, transfect and let cells recover...selection of transfection buffer and electroporation pulse program to achieve high transfection efficiency...delivered into cells; for each cell type, the transfection/delivery step needs to be optimized to achieve.... To calculate the tagging efficiency in the transfected cells, we count the number of tag-positive cells...

  15. pSiM24: Simplifying Plant Genetic Engineering

    Type
    Blog Post
    Published
    Sept. 29, 2015, 2:30 p.m.
    ...tumor-inducing” or Ti plasmid, which allows it to transfer genetic material into the host plant genome. Scientists...Scientists have worked to optimize this system for gene transfer, studying the stability of modified Ti plasmids...borders specify the region of DNA that will be transferred to the plant, similar to viral vectors’ terminal...binary vectors are quite large (>10 kb.) Since transformation efficiency is inversely proportional to size...size, and many plant scientists want to transfer large genes, smaller binary vectors with a larger cloning...ORI, they achieved much higher DNA yield and transformation efficiency in both types of bacteria. They ..., respectively, making it ideal for large gene transfer. The M24 promoter provides constitutive, high-...

  16. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    Published
    Oct. 11, 2018, 12:30 p.m.
    ...number of transformants); the greater the number of inserts, the lower the number of transformants. To compensate...containing the transformants onto the selection plate. Figure 2 shows representative transformation plates obtained...background by antibiotic selection. Screening of transformants by colony PCR confirmed the insertion of the...volume of the 1 ml outgrowth spread on each transformation plate can be manipulated to result in appropriate...assembly was easily achievable with high levels of transformants and low backgrounds – so much so that there ...improvements (5- to 12-fold increases in the number of transformants while maintaining high levels of correct assemblies...

  17. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    Published
    Nov. 5, 2024, 8:32 p.m.
    ...only have to worry about cloning your transfer plasmid. The transfer plasmids have different packaging capacities...AAV and lentivirus requires three plasmids — a transfer plasmid containing your gene of interest plus ...in mammalian cells is relatively quick. After transfection, HEK-293T cells start producing virus within...systems, it is standard to do only one passage (transfection). This differs from typical insect cell preparations... baculovirus requires only two plasmids — your transfer plasmid and the baculovirus construct, called ...between insect and mammalian systems. Baculovirus transfer plasmids have a much larger packaging capacity...Ararat, M., & Graham, F. L. (2002). Preferential transformation of human neuronal cells by human adenoviruses...

  18. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    Published
    April 13, 2021, 1:15 p.m.
    ...in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF into the BL21... This would result in the lack of recovered transformants. Our speculation motivated us to update the ... sequence in pEcgRNA. Next both plasmids are transformed in E. coli with the donor DNA for editing to ...DE3) that resulted in a lower efficiency of transformation for pTargetF series plasmids. Fortunately, ...plasmids (pDonor and pQCascade) with 4 rounds of transformation to complete the N-acetylglucosamine strain ... to construct 13 plasmids with 14 rounds of transformation in 30 days. Figure 2: (A) MUCICAT ...integration frequency. (C) Increasing rounds of transfer on transposition-inducing agar lead to higher ...

  19. Viral Production at Addgene

    Type
    Blog Post
    Published
    Oct. 24, 2023, 1:15 p.m.
    ...of Pennsylvania Vector Core. Transfections are performed using the transfer plasmid, a plasmid encoding...PCR with primers targeting the transfer plasmid used in the transfection. PCR products are analyzed by ...taken offline and discarded. Confirmation of the transfer plasmid Addgene uses a rigorous barcode matching... matching system and PCR-based confirmation of transfer plasmids used in Addgene’s viral production. Barcoding...After production, we confirm the identity of the transfer plasmid in the viral preparation by performing...performing PCR against unique regions of the transfer plasmid.   Lentivirus resources Browse lentiviral preps...

  20. New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression

    Type
    Blog Post
    Published
    Jan. 24, 2023, 2:15 p.m.
    ...transient cotransfection of the mARG plamsids into HEK293T cells using a chemical transfection reagent ...proteins. Excess gvpA plasmid added into the transfection mixture in HEK293T cells gave drastic improvement...These plasmids can be used directly in transient transfection in rtTA+ cell lines (such as HEK293-TETOn cells...they can also be integrated into the genome by cotransfecting them with a plasmid expressing PiggyBac transposase...saturate expression. When engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac ...reagent (e.g. PEI-MAX). Simply cotransfect a mixture of 1248 ng of CMV-gvpA-IRES-BFP plasmid and 954 ng CMV-...visible under phase contrast starting day 2 after transfection.     Fig: 2: Expressing mARGAna in MDA-MB...
Showing: 41 - 60 of 363 results