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We narrowed to 457 results for: nsf

Showing: 101 - 120 of 457 results

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

    Type
    Blog Post
    ...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...

  2. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    ...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...

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

    Type
    Blog Post
    ...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 ...

  4. Viral Production at Addgene

    Type
    Blog Post
    ...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...

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

    Type
    Blog Post
    ...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...

  6. Plasmids 101: TOPO Cloning

    Type
    Blog Post
    ...planning to transform right away OR you can store the reaction at -20C overnight. 4. Transform TOPO Cloning...researcher to perform blue-white screening after transformation - self joining of the vector ends results in... minutes will not significantly improve the transformation efficiency).  5. Select and Analyze 10 White...add will depend on whether you are planning on transforming your reaction into chemically or electro-competent...that you exceed the 5 minute time limit (lower transformation efficiencies have been reported with longer...antibiotic-containing plate prior to plating your transformation may allow you to see colonies within 8 hours...

  7. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    ...contamination from uncut plasmid. 4. Transformation Transform your ligation reaction into your bacterial...perform, at minimum, two transformations after a ligation: 1. Control Transformation containing the ligation... how many colonies you should expect in the transformation due to background re-circularization and contamination...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent...ligation mixture with backbone alone; 2. Transformation containing ligation mixture with insert and backbone...conduct a positive control to ensure that your transformation worked. You should also verify that you are...

  8. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

    Type
    Blog Post
    ...students first transform the S. cerevisiae met deletion strains and then isolate transformed strains on media...organism into overexpression plasmids. Students next transform deletion strains from the reference organisms ... (7). In our class research project, students transform S. cerevisiae met deletion strains with overexpression...expression of the plasmid-encoded MET gene allows the transformed strain to grow in the absence of methionine (... to select yeast that have been successfully transformed with the plasmids, because the S. cerevisiae ...of MET/Met genes from the plasmids allows the transformed strains to grow in the absence of methionine....

  9. Cloning Mammalian Cells with the Agarose Method

    Type
    Blog Post
    ...growth rate/transfection efficiency is not known, this is easily achieved by splitting transfected cells into...specific cell line, and its transfection efficiency. We often trypsinize the transfected cells on the day after...after transfection into plates containing selection agent at different densities, then dispose of the plates...under-expressing clones. If GFP-encoding cDNA is co-transfected at a favorable ratio to the gene of interest ...expression characteristics from a population of transfected clones. The technique is applicable to a wide...

  10. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    ..., expressing Ad E1A and E1B, can be transfected with your transfer plasmid, carrying your gene of interest... performed 12–18 hours post-transfection to help remove toxic transfection reagents and keep the cells...helper plasmids, commonly called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps...cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and then collect the supernatant...Parts of the AAV Packaging PlasmidParts of the AAV Transfer PlasmidAAV Variables That Matter AAV protocols...

  11. CRISPR 101: Ribonucleoprotein (RNP) Delivery

    Type
    Blog Post
    ...site of interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes...this method:     Cells must be amenable to transfection or viral transduction     Appropriate promoters... often be used in cells that are difficult to transfect, such as primary cells. Using RNPs can also alleviate... are detectable at high levels shortly after transfection, and are quickly cleared from the cell via protein...template. Other techniques, such as lipid-mediated transfection (B in the figure above), are in the early stages... this are Polyethylene Glycol (PEG) mediated transfection of protoplasts and biolistic bombardment of ...CRISPR studies in cells that are difficult to transfect or transduce. While using Cas9-gRNA RNPs may require...

  12. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    ... lines that are highly transfectable.  The amounts of each plasmid transfected into cells are also extremely...each encoded on separate plasmids and are co-transfected into cells alongside a plasmid encoding the minigenome...expression levels of each gene by altering the amount transfected (see Technical Tips). Minigenome replication ...VP30, L, and the minigenome) are efficiently transfected into cells. All of these components are necessary... format). However, since plasmid quality and transfection efficiency may vary between labs, initial titration.... This can be accomplished in multiple ways: transfecting with an additional plasmid containing the T7...

  13. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    ... the pooled library is transformed into E. coli for amplification. Transformed E. coli are allowed to ...sequencing to confirm library representation. Transfection of the DNA pooled library and appropriate packaging...Addgene.) This small aliquot is not enough to transfect producer cells, so your first step will be to ...library. This is similar to amplifying a plasmid: transform E. coli with the pooled library DNA, allow the...red.    Unlike a standard single plasmid transformation, though, a pooled library has hundreds to millions...in Lenti-X 293T cells using a polyethyenimine transfection protocol. I also recommend searching PubMed ...

  14. Tips and Tricks for Using Golden Gate Modular Cloning (MoClo)

    Type
    Blog Post
    ... after each transformation and/or use different E. coli strains with higher transformation efficiency....robot, you can set it up to automate the reagent transfer and the restriction–ligation reactions in a 96...colonies) after each consecutive assembly and transformation round, due to the decrease in ligation efficiency...’re cloning in high-throughput, we recommend transforming into chemically competent cells in a 96-well...have what look like positive colonies on your transformation plates. However, even though the MoClo system... always screen multiple colonies after each transformation. This could be as few as 1–2 colonies if you...

  15. Tips for Using FRET in Your Experiments

    Type
    Blog Post
    ...Bioluminescence Resonance Energy Transfer CRET= Chemiluminescence Resonance Energy Transfer    Interesting Articles...FRET, aka Fluorescence (Förster) Resonance Energy Transfer, technique which allows the detection of molecules...distance-dependent physical process by which energy is transferred non-radiatively from an excited molecular fluorophore...higher than the signal generated by the energy transfer. On the contrary, if you choose a pair that are...cross-talk but you also don’t have any resonance transfer. You have to find a balance between FRET efficiency...RB, Periasamy A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein...

  16. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...single gene. The gRNA expressing cells were then transfected with a Cas9-expressing plasmid to generate a ...genome engineering and synthetic biology have transformed the way scientists create new traits in bacteria...inhibitors of epigenetic modifiers like DNA methyltransferases or histone deacetylases. Unfortunately, these...dCas9) with the catalytic domain from DNA methyltransferase, DNMT3A. Co-expression of this modified Cas9...homologous recombination, and can be difficult to transform. To increase the genetic toolbox for industrial... and cloned into a third generation lentiviral transfer vector. Each gene has been FLAG-tagged for convenient...luciferase. The bait and prey plasmids are co-transfected and proteins are immunoprecipitated from the ...

  17. Why Do I Need an MTA Anyway?

    Type
    Blog Post
    ...Uniform Biological Material Transfer Agreement (UBMTA) for its material transfers. The UBMTA was drafted by... has time for paperwork? Here’s why a Material Transfer Agreement (MTA) matters: Science thrives on collaboration...universities and institutions require them for material transfers. When Addgene entered the plasmid sharing space...receiving institution. Therefore, many technology transfer officers are familiar with the UBMTA and are happy...  material transfer agreement...

  18. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    ...activation p300 acetyltransferase dCas9 fused to the catalytic domain of p300 acetyltransferase increases levels...transcription, also includes light-regulated histone methyltransferases and deacetylases. CRISPR and epigenetics ...delivery systems. Transcriptional repression DNA Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš’ lab has deposited...repression. Created with BioRender.com. DNA Methyltransferase MQ1 Margaret Goodell's lab has deposited pcDNA3.1...Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase from the prokaryote Mollicutes spiroplasma... Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers...

  19. Brain Armamentarium

    Type
    Collection
    ...specimens. These technologies have the potential to transform neuroscience research by bringing the precision...

  20. Plasmids 101: NGS Quality Control for Pooled Libraries

    Type
    Blog Post
    ... seems straightforward: E. coli bacteria are transformed with the library DNA, they grow and replicate... Each Addgene library has its own depositor transformation and amplification protocol, but the overall...Maintain representation of the library during transformation First, use enough DNA and competent bacteria...and we use electroporation to ensure a good transformation efficiency. For a typical library, Addgene ...growing the cells on solid plates, where each transformant forms its own colony, uninfluenced by the growth...concern, but Addgene still recommends plating the transformation mixture on solid media. The colonies are then...
Showing: 101 - 120 of 457 results