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We narrowed to 789 results for: mpl

Showing: 181 - 200 of 789 results

  1. Plasmids 101: Terminators and PolyA signals

    Type
    Blog Post
    Published
    March 31, 2016, 2:30 p.m.
    ...structure of the hairpin-DNA complex is thought to destabilize the transcription complex, initiating cleavage ...transcript. Rho-dependent terminators are not usually employed in plasmid-based expression systems, so these ...100% efficient at halting transcription of the template and initiating the desired cleavage event, although...polymerase terminates differently. Polymerase III, for example, relies on a specific sequence and RNA secondary...termination factor dependent, Polymerases I and II employ different mechanisms to terminate transcription...mechanism, whereas Polymerase II termination is more complex and involves two RNA polymerase-associated proteins... care (4). For this reason, viral vectors often employ other non-poly(A) transcript-stabilizing and nuclear...

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

    Type
    Blog Post
    Published
    Oct. 23, 2018, 11:49 a.m.
    ...transfer plasmid. The good news is there’s a few simple ways to deal with this issue. One way is to grow...helps me figure out if I have enough plasmid to complete my experiment. AAV is usually the limiting reagent...concentrate the purified virus. This process can be completed in one long day, or the virus can be stored at...particles that are present in an AAV prep. AAV samples are first DNaseI digested to remove any residual...technology or a TaqMan primer/probe set can be used and samples are quantified by comparing to an AAV standard ... plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR...AAV, remember to account for the dilution of the sample for the DNAseI digest. Use good sterile technique...

  3. Google Forums Round Up: First Impressions of NgAgo

    Type
    Blog Post
    Published
    Aug. 4, 2016, 2:30 p.m.
    ...inexpensive and can be 5’-phosphorylated in house using a simple PNK reaction or can be purchased 5’-modified from...probably while it is being synthesized) can form a complex with an ssDNA guide. Therefore, researchers must...inject cells with in vitro preassembled Cas9/gRNA complexes or with guide RNA- and Cas9-expression plasmids... an optimal distance from the target site. For example, NGG may not be present in AT-rich regions of the...same method used by Gao et al, 2016 (1). View the Complete and Up-to-Date Results of Pooran's NgAgo Survey..., leaving a small amount of active NgAgo/ssDNA complex in the cell. As proposed in the original article...protein molecules manages to form a functional complex with ssDNA leading to inefficient downstream editing...

  4. PITChing MMEJ as an Alternative Route for Gene Editing

    Type
    Blog Post
    Published
    Feb. 23, 2016, 3:30 p.m.
    ...about a month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction...double-stranded break. HDR copies the sequence from a repair template with flanking sequence homology for error-free...systems, and NHEJ will be favored even if a repair template is present. HDR also presents a cloning dilemma...required by MMEJ can easily be added via PCR amplification. Given the inefficiency of HDR for knock-ins...strategy looks very similar to HDR, but it is accomplished using much smaller regions of homology, which...strategy looks very similar to HDR, but it is completed using much shorter sequence homologies. Abbreviated...Select for puromycin resistant cells Step 5: PCR amplify and sequence the locus to verify correct GFP-puro...

  5. Genome-wide Screening Using CRISPR

    Type
    Blog Post
    Published
    Aug. 18, 2015, 2:30 p.m.
    ...over previous genome editing techniques is its simplicity and versatility. CRISPR/Cas9 consists of two ...jar of jellybeans, a pooled CRISPR library is a complex mixture. Pooled libraries are composed of many ...Pooled lentiviral CRISPR libraries (referred to simply as “CRISPR libraries”) consist of a heterogeneous...for experimental use. Thus, the first step is to amplify your library to a concentration that is sufficient...next-generation sequencing to check the quality of your amplification. If you've obtained a ready-to-use lentiviral...sorting and screened for a particular phenotype. For example, mutant cells can be used in drug screens to identify.... Figure 2: The CRISPR library must be amplified (A) to be used to generate lentivirus (B). Cas9...

  6. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    Published
    May 28, 2024, 1:45 p.m.
    ...carry their emission peaks within their names, for example, BV421 and RB545. Fluorophores can also come in...recognize a couple of fluorophores from Figure 1. For example, the chart tells us that PE and PE-Cy7 are "very...en-us/resources/bd-spectrum-viewer.   Another example APC is a "bright" fluorophore when excited by the... of neighboring colors can and do overlap. For example, APC overlaps noticeably with both BV605 and Alexa...antibody-conjugated labeling. Even though the two compounds are completely different in nature (eGFP being a protein and...build your first panel! Designing a Panel In this example, I will describe a basic panel (Table 2, Figure...much higher than CD45 expression, this could, for example, point at issues with the double-expression vector...

  7. Capturing a Moment with Marc Zimmer’s “The State of Science”

    Type
    Blog Post
    Published
    July 30, 2020, 1:15 p.m.
    ...years ago. Teaching by example No examination of the state of science would be complete without the inclusion...attempts to answer it in five paragraphs, with examples. If that sounds ambitious, that’s because it is...non-scientist friends and family is Part Five, called simply “Bad Science.” Zimmer kicks off the section saying...provides several. In Part Three of the book, called simply “Old Science,” he describes the now infamous tale...Four, “New Science,” Zimmer tackles more recent examples of scientific advances, and posits the way science... much less familiar with as a biologist--as an example of “big science,” a massive, expensive project ...CRISPR of course address some of the ethical implications of using these technologies, but also the rapidity...

  8. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    Published
    July 27, 2023, 1:15 p.m.
    ...referred to as “complex retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, ... we will mostly use the gamma-retroviruses as a simple spokesmodel for the whole family; however, you ...beginning to realize that the situation is already complex enough that a deep understanding of retroviral ...polymerase gene may seem like a mystery. But it’s simple. The RT protein is made by a separate set of instructions... a tool is done, and the transgenic genome will simply express from whatever promoters were included in...Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiological Reviews, 56(3), 375...Initiation of synthesis with 70 S viral RNA as template. Journal of Molecular Biology, 79(1), 163–183....

  9. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    Published
    Dec. 22, 2022, 2:15 p.m.
    ... will guide the cell to use the donor DNA as a template for repair, which will result in the incorporation... knock-in! So how does the donor get used as a template for repair? Read on to find out! Homology-directed...basis of the HDR pathway relies on repair from a template molecule, which is usually an endogenously available...and uses the ends of the ssDNA to find a repair template. The highest HDR efficiency is observed when inserts...making the cut off essentially as part of the templated DNA. Check out our blog post on increasing HDR...To make sure your donor molecule is used as the template, you need to incorporate homologous sequence to...sister chromatid is likely to exist as a repair template. To maximize HDR events, ensure cells are actively...

  10. Save Time with Transient Plant Leaf Transformations

    Type
    Blog Post
    Published
    July 25, 2019, 12:03 p.m.
    ...surface) of the leaf. This is a simple and easy technique to implement. Success might vary between experimenters...tobacco, Agrobacterium infiltration into leaves is a simple and efficient way to overexpress a gene and evaluate...your experiment (de Ruijter et al., 2003). For example, we did our initial transformations with the GUS... on transformation success. Some reporter gene examples are below. The GUS gene (β-glucuronidase) with...temporal reporters to measure promoter activity, for example. Useful positive controls could include: pSB96 ...expression from Agrobacterium, but not the plant. For example, we have seen strong expression of the GUS gene... Li, Jian-Feng, et al. "The FAST technique: a simplified Agrobacterium-based transformation method for...

  11. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    Published
    March 12, 2019, 1:37 p.m.
    ...chalk talk? On the surface, a chalk talk is quite simple. It's you, a dry erase board, some markers (chalk...question, not a vague goal or a "what" question. For example, "we want a better understanding of the Z pathway...about the time and resources required to feasibly accomplish the aims and clarify what type of aim you are...scientists outside your field, and interesting. For example, “there is a significant need for healthy livers...explain why it’s significant and innovative. For example, "while it is possible to grow liver stem cells...exist to solve the problem. What will you have accomplished in 5-10 years? You need to be able to describe...learned and/or now be possible to do? Research accomplishments are remembered for the problem solved, not ...

  12. Supporting Reproducibility with a Connected ELN

    Type
    Blog Post
    Published
    Jan. 17, 2019, 1:38 p.m.
    ...in which the two guiding principles have been implemented by providing support for connectivity to external...Without such references the record of research is incomplete, making it difficult or impossible to reproduce...Github and Gitlab, and science-specific tools, e.g. sample management apps, colony management software, and...the record of research captured in the ELN is incomplete. Thus, to support reproducibility an ELN should...provide a direct boost to reproducibility. For example, it’s possible to import Word documents into RSpace...possible to link between various documents. A second example is the integration between RSpace and Slack, illustrated...subsequently reproduce the research. A third, powerful example is the integration with protocols.io, the protocol...

  13. pCXLE toolkit: Efficient episomal plasmid-based method to reprogram peripheral blood cells to iPSCs

    Type
    Blog Post
    Published
    Dec. 14, 2017, 2:08 p.m.
    ... OriP and induces amplification of the episomes concurrently with DNA amplification in the host cell. ...blood sample, in the best-case scenario [7]. In the clinical setting where patient blood samples are limited...especially if used to make clinical grade hiPSCs. For example, the CytoTune™-iPS 2.1 Sendai Reprogramming Kit...persistence of residual viral material [6]. The complete removal of the residual viral particles and establishment... in the presence of these two factors are not completely understood, the addition of an shRNA for P53 ...grade hiPSCs from easily accessible patient blood samples. All pCXLE vectors listed below in Table 1 are ...

  14. Changing Labor Laws Bring Increased Postdoc Wages

    Type
    Blog Post
    Published
    Oct. 25, 2016, 2:30 p.m.
    ...for implementation was set for Dec 1 2016. In order to be exempted from the need to pay employees overtime...submitted letters to the Department of Labor (for example here and here) pushing for exemptions for postdocs...institutions doing? Institutions are now preparing to comply with the changes to the law on Dec 1st, according...centrally administered, which leads to many complications, including the inability of some institutions...that they are raising salaries as their website implies. These 5 organizations, according to the NSF data...they're thinking about applying to, are doing - for example, your benefits may be changing. Please also feel...wide consensus of academic stakeholders. There was ample opportunity to accept these recommendations, and...

  15. Data Freedom: The Expansion of Data Sharing in Research Publications

    Type
    Blog Post
    Published
    Aug. 5, 2014, 6:51 p.m.
    ...some data, etc.  The firestorm that followed implementation of the policy comprised of numerous objections...raw data requires huge amounts of storage. For example, mouse behavioral geneticists may track the motion...although it might be argued various authors have simply chosen to submit elsewhere). The majority of the...numbers of figures such that many have multiple, complex panels. PLOS, itself, has not finalized its definition... paper are in scope. For images, the number of examples supporting the published material is a factor ...types? And how long should data be archived? Simplifying data access Some of the above mentioned issues...collections and interpretation of data, publishing complete data, rather than just a single projection of ...

  16. Inside Addgene's 10 Year Anniversary Gala

    Type
    Blog Post
    Published
    July 3, 2014, 3:48 p.m.
    ... relatively simple, changed the course of scientific research. What was once a complicated process of ...and requesting scientists, all of the Addgene employees, the Addgene board members, vendors, Addgene supporters...prototype of an interactive game that Addgene employees created and designed for the Hall of Human Life...playing all night was hand-picked (like our plasmid samples!) by Addgenies. If you were there, you might have... on sliders, hot dogs and a mashed potato bar (complete with all the fixings), as well as plenty of delicious...of requesting and sharing plasmids has become simplified through Addgene. Looking back, Addgene’s journey...

  17. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

    Type
    Blog Post
    Published
    Sept. 1, 2020, 1:15 p.m.
    ...then following transduction, the partial genomes complement each other to restore the full-expression cassette...strategy for increasing the size of the AAV donor template for CRISPR-mediated homology directed repairs ...co-transduced with multiple copies of both vectors. Examples of use: Halbert et al., 2002 used an overlapping...outperform single oversized AAVs (Hirsch et al, 2013). Examples of use: Lai et al., 2005 used a trans-splicing... the accumulation of such truncated proteins. Examples of use:  Ghosh et al., 2008 used a hybrid split...homologous recombination approach delivers large donor templates using two sequential homology directed repairs... the gRNA target site that mediated the donor template’s integration so that the site is reconstituted...

  18. Plasmids 101: Expressing shRNAs for RNA Interference

    Type
    Blog Post
    Published
    June 17, 2025, 1:15 p.m.
    ... by a ribonuclease called Dicer. In a great simplification of the process, Dicer chops off the loop of...create pri-miRNA, which is then processed by a complex called Drosha to pre-miRNA. miRNA plasmids are ...RNA duplexes bind to the RNA-induced silencing complex (RISC). After binding to RISC, the RNA duplex is...t always cleave the mRNA — the bound miRISC can simply block translation machinery. The composition of...typically range from 19–22 nucleotides and are 100% complementary to their target sequence. However, you can’t... day. You need to include both sides of the complementary RNA duplex and the hairpin loop sequence (Figure...action, and degradation of RNA-induced silencing complex. Molecular Cell, 82(1), 30–43. https://doi.org/...

  19. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    Published
    Aug. 23, 2018, 12:05 p.m.
    ...to the popularity of GFP including (i) fast and complete maturation to functional, fluorescent protein ...endogenous fluorescent co-factors can be used. Examples include the LOV domains that bind flavin (Buckley... pKa, which is around 6.0 for GFP. This number implies that at pH 6 only 50% of the available green fluorescent...under acidic conditions, with pKa values below 4. Examples include mTurquoise2 (pKa = 3.1), tagRFP (pKa =...diameter of about 2.4 nm (Hink et al., 2000). The complete beta-barrel is necessary for its fluorescence ...specifically and with high affinity to fluorophores. Examples are the FlAsH tag, the His tag and several others... proteins are also a better choice for thicker samples. Another solution to avoid autofluorescence is ...

  20. Build Your CRISPR Vocabulary

    Type
    Blog Post
    Published
    July 2, 2024, 1:15 p.m.
    ...Palindromic Repeats. That contrast of simplicity and complexity is reflected in the biology, too. CRISPR..., and the spacer sequence is used to target complementary DNA sequences. In laboratory settings, an sgRNA...whose sequence is not checked. Assuming full complementarity, its sequence matches the guide sequence. PAM...into a single strand that folds back on itself, simplifying the CRISPR-Cas9 system for laboratory applications...either crRNA or sgRNA). Cas9 requires perfect complementarity in this region between the guide and target...post on the structural biology of Cas9. Want a simpler guide to keep at your desk? We’ve got a CRISPR ...
Showing: 181 - 200 of 789 results