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  1. Addgene Receives NIH BRAIN Initiative Grant to Create Open-Access Recombinant Antibody Resource

    Type
    Blog Post
    Published
    May 25, 2021, 2:29 p.m.
    ...received a grant from the National Institutes of Health that will address this problem.The grant, which is...within the next year. NIH grants and Addgene’s mission This is the first NIH grant that Addgene has received... of California Davis School of Medicine on this grant. Dr. Trimmer has created thousands of high-quality...NIH’s vision and support for this project. This grant will allow Addgene to further our mission of scientific...working with the community to create a fully transparent resource where researchers can easily share, ...project will be supported by a NIH BRAIN Initiative grant (NS119916). The content of this article is solely...P, Frenzel A, Wong J, Koh XY, Eng H-Y, Lane D, Lefranc M-P, Clark M, Dübel S (2018) When monoclonal antibodies...

  2. Walking beside Academia and giving it a high five: My transition from graduate work to a position at Addgene

    Type
    Blog Post
    Published
    July 2, 2015, 8:37 p.m.
    ...plasmid page here). Now I spend much less time wrangling bacteria and instead help Addgene fulfill its ...better and make scientists’ lives easier. Why transition to Addgene? So why did I join Addgene? Was I ...easier. I’m hoping my story demonstrates that transitioning from academia doesn’t have to mean abandoning...management, information distillation, etc) are transferable to many different fields. If you start thinking...

  3. Addgene-Seeding Labs plasmid grant round 3: microbial drug resistance and biofilms, antidiabetic plant components, visualizing brain injury - and a glowing robot fish

    Type
    Blog Post
    Published
    Nov. 27, 2018, 2 p.m.
    ...fluorescent protein transcription in response to river contaminants and transform these plasmids into ...Addgene-Seeding Labs Plasmid Grant Check Out Addgene-Seeding Labs Plasmid Grant Awardees in 2016 and 2017... specifically, the Addgene-Seeding Labs plasmid grant provide plasmids to these researchers. In the third...third round of the Addgene-Seeding Labs plasmid grant we are excited to introduce the 5 awardees and their...into a bacterial strain. The sensor will be transported inside a small 3D printed robot that will “swim” ... diverse groups of scientists working in a wide range of research fields and geographical locations. In...see how all of the Addgene-Seeding Labs Plasmid Grant awardees' projects progress. Subscribe to the Addgene...

  4. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    Published
    Jan. 26, 2016, 3:30 p.m.
    ...course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and AAV can be used...Thakore PI, Moreb EA, Rivera RM, Madhavan S, Pan X, Ran FA, Yan WX, Asokan A, Zhang F, Duan D, Gersbach CA...WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, ...: 25692716. PubMed Central PMCID: PMC4335351. 9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...) to remove mutated exons from the dystrophin transcript. Unfortunately, the oligonucleotides only modestly...in one experiment, they found the exon-skipped transcript constituted 59% of total dystrophin mRNA, similar...low to no off-target activity at the ten highest-ranking predicted off-target sites. Each paper characterizes...

  5. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    Published
    March 13, 2014, 4:08 p.m.
    ...probably have better specificity. A good reference is Ran et al. (2013). As for target selection, we usually...well in 24-well plate. A good example/reference is Ran et al. (2013). In the case where you have intact...https://doi.org/10.1534/g3.112.005439  Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N...., 93–101. https://doi.org/10.1128/mcb.18.1.93  Ran, F. A., Hsu, P. D., Lin, C., Gootenberg, J. S., Konermann...1389. https://doi.org/10.1016/j.cell.2013.08.021  Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott...mutation, the best HR template for transfection is probably a single stranded DNA (ssDNA) oligo which usually...positively transfected. Alternatively, you can use a selection marker to select transfected cells (for...

  6. Overcoming the AAV Size Limitation for CRISPR Delivery

    Type
    Blog Post
    Published
    Sept. 16, 2020, 1 p.m.
    ...874 . https://doi.org/10.1038/nmeth.3993  Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu.../doi.org/10.1038/s41586-019-0908-x  Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani...949. https://doi.org/10.1016/j.cell.2014.02.001  Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of ...prime editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge...split AAVs. In general split AAVs break a large transgene in two pieces and package each piece into an individual... individual AAV. When a cell is transduced by both AAVs, the full length gene and/or protein is reconstituted...

  7. Scientists Map the SARS-CoV-2-Human Interaction Network

    Type
    Blog Post
    Published
    April 9, 2020, 1:15 p.m.
    ... two of the remaining viral proteins. Only Nsp11 ran bigger than expected, and Orf7b gave a lot of background...received so many requests (175 and counting) that we ran out of FedEx supplies at one point and had to go ...right) from the University of California, San Francisco. Manon Eckhardt and Melanie Brewer are on the ...

  8. Interview with Bart Newland, Addgene’s Newest Board Member

    Type
    Blog Post
    Published
    Aug. 3, 2021, 1:15 p.m.
    ...For the last 20 years, I was a patent counsel and ran a group at two fairly large companies, Genzyme and...What is your day-to-day like? BN: I've made the transition now to being general counsel of a small start-up...

  9. Keeping Up With CRISPR/Cas9

    Type
    Blog Post
    Published
    March 11, 2014, 5:55 p.m.
    ...limitations of the Cas9 complex," said study author F. Ann Ran, a graduate student in Feng Zhang's lab, in a press..., in the labs of Chase L. Beisel and Rodolphe Barrangou, had applied the CRISPR/Cas9 system to developing...

  10. Your Top Requested Plasmid in 2016!

    Type
    Blog Post
    Published
    Jan. 11, 2017, 8:47 p.m.
    ... Publication describing them here. References 1.Ran, F. Ann, et al. "Genome engineering using the CRISPR-Cas9...plasmid post transfection. The 2A "self cleaving" peptide causes the ribosome to translate Cas9-2A and ...component of most CRISPR systems and generates double strange breaks at target sites 5' of the 5'NGG3' PAM sequence...backbone. This particular backbone can be used to transfect a variety of mammalian cells and can be modified...are some additional features. The first and most imporant is that, upstream of the Cbh promoter, this plasmid...can be used to test for SpCas9 expression post transfection by western blot. Overall, this is a versatile...

  12. Addgene at the ASM Microbe 2016 Conference

    Type
    Blog Post
    Published
    July 7, 2016, 2:30 p.m.
    ...great place to catch up with former colleagues! I ran into past ASM President Tim Donohue (see below) on... meet hundreds of the over 11,000 conference registrants, including scientists from six continents who...

  14. An Addgene Summer Internship

    Type
    Blog Post
    Published
    Aug. 8, 2024, 7:22 p.m.
    ...two weeks, we were able to obtain eight clones. We ran digested and undigested samples of the clones on ...and I wouldn’t want it any other way. Toj Toj-Villagran is a rising senior in high school who just completed...

  16. Celebrate National Book Month with Addgene

    Type
    Blog Post
    Published
    Oct. 3, 2019, 12:47 p.m.
    ...disliked, some that everyone loved, and some that ran the spectrum. I recently presented Kurt Vonnegut’...without compromising on the relevant science."     - Randall Carter, Contracts Administrator Inferior: How Science... Non-science favorites  The Mistborn series by Brandon Sanderson "The Mistborn series is the first 3 books...Associate Director of Quality Control Jonathan Strange & Mr. Norrell by Susanna Clarke "A quasi-historical...Vonnegut’s Slaughterhouse-Five, and the responses ranged from “one of my favorite books” to “I barely got through...

  17. Advice for Scientists Starting a Lab

    Type
    Blog Post
    Published
    April 29, 2014, 1:43 p.m.
    ...wanting to know our latest results as soon as the gels ran.” Dr. Ellis tries to lead his team as a scientists.... “Being a PI is more than just experiments and grant writing. You have to enjoy the mentoring role.” ...emphasis on genetic engineering, sequencing, and translation of results to companies.” Take home message Starting...

  18. History of CRISPR Cas - A tale of survival and evolution

    Type
    Blog Post
    Published
    Nov. 25, 2013, 10:12 p.m.
    ...Davidson AR.  Nature. 2013 Jan; 493, 429–432. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...and are not translatable to the other CRISPR subtypes. Still, the large and dynamic range of CRISPR systems...well-suited for various innovative biological and translative technologies. Addgene has empowered researchers...http://en.wikipedia.org/wiki/File:Crispr.png Barrangou R, Horvath P. Science. 315, 2007 Jan; 1709–1712...CRISPRdb. Date: 2013-09-18. Makarova K, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, Moineu...

  20. CRISPR 101: Non-Homologous End Joining

    Type
    Blog Post
    Published
    April 16, 2015, 3:45 p.m.
    ...842 . https://doi.org/10.1016/j.molcel.2014.08.002 Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, ... single strand break; when two such breaks are introduced near each other, in opposite strands. This “...Cas9 can be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses....org/10.1371/journal.pgen.1000683 Bétermier M, Bertrand P, Lopez BS (2014) Is Non-Homologous End-Joining...Giovannangeli C, Jasin M, Brunet E (2014) Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous.../doi.org/10.1016/j.cell.2013.08.021 Waters CA, Strande NT, Pryor JM, Strom CN, Mieczkowski P, Burkhalter...

  21. Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens

    Type
    Blog Post
    Published
    Feb. 24, 2015, 7:50 p.m.
    ...complex with guide RNA and target DNA. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani... sgRNAs for each transcription start site in 15,977 human genes. The transcriptional repressor (CRISPRi...SAM is an engineered protein complex for the transcriptional activation of endogenous genes and it consists...The next insight was to assemble a synthetic transcription activation complex consisting of multiple distinct...together synergistically, modeled after natural transcription activation processes. Konermann and her colleagues...consequences of repressing or inducing individual transcripts. The activator (CRISPRa) sgRNA library uses the...CRISPRi) library contains 10 sgRNAs for each transcription start site in those 15,977 human genes. Weissman...

  22. Reaching out to China: Canton Nucleic Acids Forum (CNAF) 2015

    Type
    Blog Post
    Published
    Dec. 1, 2015, 3:30 p.m.
    ...fragments of these transcripts. I remember when small RNA was just something we ran off the bottom of ...’t give tRNAs much thought. High throughput transcriptome sequencing methods including work done by the...hypothesized that life started with RNA and a simple cell membrane. His recent work shows that they are making fairly...knew…I had barely heard of these). These single stranded, covalently closed RNAs seem to be conserved in...

  23. Story of a SynBio Startup: RevBio's Epiphany (or Lack Thereof)

    Type
    Blog Post
    Published
    June 5, 2014, 3:32 p.m.
    ...that produce GMO food plants. When a press piece ran on the detector plants, we expected to see that same...below. We’re happy to answer questions about this transition (so far) and what we’ve learned.  Thank you ...

  24. Design Tips for Prime Editing

    Type
    Blog Post
    Published
    Jan. 23, 2025, 2:15 p.m.
    ...doi.org/10.1038/s41587-021-01039-7 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I....nicking of the unedited strand and encourage the cell to use the edited strand as a template during DNA...on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing guide RNA (pegRNA). The ...) complementary to the genome and a reverse transcriptase (RT) template including the desired edit. ...although sequences with G/C content outside this range can still be optimized (Anzalone et al., 2019). .... Test different lengths of reverse transcriptase template, starting with about 10–16 nucleotides. For...pegRNA primer binding site (PBS) and reverse transcriptase template (RTT). Created with BioRender.com. ...

  25. DNA Purification Without a Kit

    Type
    Blog Post
    Published
    Feb. 11, 2020, 2:15 p.m.
    ...just make their own buffers when the kits buffers ran out. Homemade buffers performed the same as those...rather a glass powder slurry that has a milk-like appearance. To make DNA stick to the glass particles in ...the Patterson Protocol was shown to purify DNA ranging in size from 100 bp to 48 kb with high yields and...could interfere with interpreting the results of transfection assays. Go forth and perform your DNA extractions...

  26. Bioinformatics at Addgene

    Type
    Blog Post
    Published
    July 14, 2022, 1:15 p.m.
    ... same output regardless of who or which computer ran the process. Another design choice we appreciated...automating the bioinformatics steps needed to transform the raw sequencing data into a complete plasmid...appropriate place in our workflow without having to translate or convert programs to a uniform language.  The...concepts and vocabulary of Nextflow so that we could translate our pipeline steps into processes and then connect...

  27. The PAM Requirement and Expanding CRISPR Beyond SpCas9

    Type
    Blog Post
    Published
    Aug. 20, 2020, 2:30 p.m.
    ... 556:57–63 . https://doi.org/10.1038/nature26155 Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...SpCas9 variants effectively double the targeting range of CRISPR/Cas9 within the human genome. Another ...known to produce off-target editing with SpCas9 (Amrani et al., 2018). When choosing a Cas9, remember to...formerly Cpf1), a nuclease that generates double strand breaks in target genes resulting in the formation...nature24049 Find plasmids from this paper at Addgene. Amrani N, Gao XD, Liu P, Edraki A, Mir A, Ibraheim R, ...018-1591-1 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F (2017) RNA editing...

  28. Binning Singletons: Tackling Conference Networking When You Don’t Know Anyone

    Type
    Blog Post
    Published
    Feb. 18, 2020, 2:15 p.m.
    ...the most out of their meetings. Amanda Gunn ran randomized coffee trials for us at ASCB|EMBO 2019, matching...within a bin as Horizontal Transfer. This comes from horizontal gene transfer, which occurs when bacteria...in Atlanta, GA, in 2018. A lot of my experiences rang true with theirs.  It was at this dinner and the...Singletons logo. Image: James et al., 2020. Help me stranger - Finding mentors for the meeting Just like analysis...

  29. CRISPR 101: Homology Directed Repair

    Type
    Blog Post
    Published
    Jan. 26, 2023, 2:15 p.m.
    ...Genome engineering using the CRISPR-Cas9 system. Ran FA, et al. Nature Protocols Nov;8(11):2281-308; (2013)...dHJs are four-stranded branched structures that form when elongation of the invasive strand “captures” and...lesion results from breakage of both DNA strands - a double-strand break (DSB). DSBs can be caused by intracellular...proteins required for strand invasion and a primer for DNA repair synthesis. The ssDNA strand then displaces ...displaces one strand of the homologous DNA donor and can template repair off the other strand; this results in...pathways: classical double-strand break repair (DSBR), synthesis-dependent strand-annealing (SDSA), and break-induced...on the crossing strand (horizontally at the purple arrows) or on the non-crossing strand (vertically at...

  30. Generating Mouse Models Using CRISPR/Cas9

    Type
    Blog Post
    Published
    July 12, 2016, 2:30 p.m.
    ...that better human health.     References Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...3.4% of founders establishing germline transmission of the transgene, this technique is still in need of ...and chimeric adults mated to produce completely transgenic offspring. Although powerful, this technology... a mouse model, and its success is not always guaranteed. To learn more about this process check out our...the Cas9 endonuclease that creates the DNA double-strand break (DSB) at the target site, and a donor oligonucleotide...

  31. A History of Genome Engineering in Popular Culture

    Type
    Blog Post
    Published
    Feb. 25, 2020, 2:15 p.m.
    ... . https://doi.org/10.1016/s0764-4469(00)01250-6 Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang...of the DNA. Finally, in 1953, Franklin along with James Watson and Francis Crick resolved the DNA double...writers have been curious about the capability of transforming the genetic code of living organisms and its...that Streptococcus pneumoniae bacteria could be transformed from one organism to the other. This is now famed...Hershey-Chase experiment. In the same year, Rosalind Franklin and her student Raymond Gosling took the first...double helix. In the 1960s, Har Gobind Khorana, Marshall W. Nirenberg, and Robert W. Holley cracked the genetic...explained how the four letters of the DNA get translated into the twenty amino acids. Once scientists ...

  32. Typing CRISPR Systems

    Type
    Blog Post
    Published
    March 18, 2025, 1:15 p.m.
    ...6105. https://doi.org/10.1093/nar/gku241 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N..... Type I systems are also employed as CRISPR transposases, by leaving out Cas3. Type I is further subdivided...complex (blue subunits) with a guide RNA (green and orange) bound to the target DNA with Cas3 (pink). Complex...promoter, allowing for easy multiplexing. The double-stranded breaks caused by Cas12 create short 3’ overhangs...paradoxically called V-K, is a CRISPR-associated transposase (CAST) that can insert large fragments of DNA...use a Cas14 enzyme, and uniquely target single-stranded DNA non-specifically. Type VI Type VI is defined...interference, as it can target and degrade select RNA transcripts, or be engineered to conduct direct RNA editing...

  33. A Needle in a Base-Stack: Cas9 Structural Biology

    Type
    Blog Post
    Published
    June 4, 2024, 1:15 p.m.
    ...10.1146/annurev-biophys-062215-010822 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I.... NUC HNH Cut DNA target strand RuvC Cut DNA non-target strand CTD (or PI) Recognize PAM...information on these regions.   Cas9’s transition from weak, random binding to precision gene editing requires...left) and transparent (right). The RNA guide sequence is extended as a single unpaired strand in the center...10 of the guide RNA, prearranged for base pairing with a complementary DNA strand. These 10 nucleotides...the non-target strand for the PAM sequence and doesn’t mind if the PAM complementary strand is mismatched...of the PAM in the non-target DNA strand (dark gray). Target DNA strand, light gray.   Once it has found...

  34. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    Published
    Sept. 17, 2024, 1:15 p.m.
    ... your membrane, run a double-membrane transfer with a psi membrane stacked behind the p membrane. If your... transfer buffer Small proteins: Over-transferring Transfer with two membranes* Psi membrane...alcohol in transfer buffer Mixed proteins: Transferring issues Transfer with two membranes* Electroblot...completely through the membrane or larger proteins not transferring completely onto the membrane. If you’re not...using two membranes during your transfer instead of one. If you see signal on both membranes, you know...large protein, you may be under-transferring.  Membrane pore size Membranes come in different pore sizes ...through the p membrane into the psi membrane, that’s a good indication that the p membrane pores are too...

  35. PiggyBac-ing Through the Genome Editing Field

    Type
    Blog Post
    Published
    May 31, 2016, 3:30 p.m.
    ...titrating the transposase to transposon ratios. High ratios of transposase to transposon result in a greater...]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The piggyBac™...piggyBac™ transposase facilitates the integration of the transposon specifically at ‘TTAA’ sites randomly dispersed... piggyBac™ transposon. These plasmids, when combined with a source of piggyBac™ transposase (available...distributor) allow you to quickly transfer a DNA sequence from the transposon vector to one of many TTAA sequences...folks at Transposagen. This post was contributed by Amar Singh and Mathew Goodwin from Transposagen What ...What is the piggyBac™ transposon? The piggyBac™ transposon was originally identified in moths in 1983, but...

  36. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    Published
    Oct. 12, 2023, 1:15 p.m.
    ...Cre-dependent transgenes can also be paired with Cre-expressing transgenic mouse lines to restrict transgene expression...with broad CNS or PNS transduction, cell type specificity, or retrograde transport in neurons. (b) Genetic...capsids or transgenes. Sensory neurons and liver appear especially susceptible to off-target transgene expression-induced...It has a similar transduction rate to AAV-PHP.eB in the brain, and a lower transduction rate in the spinal...2022). AAV-PHP.N specifically transduces NeuN+ neurons, with transduction efficiency varying from 10-70%... effectively transduces BALB/cJ, CBA/J, and FVB/NJ mice, with minimal liver transduction observed in both...effectively transduces neurons in the peripheral nervous system (PNS). It has been shown to transduce 80% of...

  37. Using AAV for Neuronal Tracing

    Type
    Blog Post
    Published
    Aug. 9, 2018, 1:04 p.m.
    ..., and enzymes for transmitter synthesis. Retrograde transport is used for transport of endocytosed material... facilitate transport: retrograde transport relies on dynein, while anterograde transport relies on kinesin...take advantage of existing transport pathways in neurons. Anterograde transport is typically used for the...mechanisms of trans-synaptic trafficking are not fully understood and may be a form of transcytosis where the...after intracellular transport without actually causing infection or leading to transgene expression within...endothelial transcytosis (Foust et al., 2009; Merkel et al., 2017), and these two modes of transport may share...optogenetically controlled transgenes, in combination with conditional transgenic mice, to reveal not only...

  38. Sleeping Beauty Awakens for Genome Engineering

    Type
    Blog Post
    Published
    June 30, 2015, 2 p.m.
    ...contains the transposase gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is ...which cuts the DNA.3.) The released transposon can now bind a strand of DNA with a TA dinucleotide (there...removal of the transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double...double strand break in the DNA and allows the tranposon to integrate. Additionally, the TA sequence is duplicated....   The Sleeping Beauty transposon system The Sleeping Beauty (SB) transposon system is one such system...perform gene transfer in vertebrates. The system is comprised of 2 components: 1) an SB transposase, the enzyme...enzyme required for catalysis of transposition; and 2) a transposon containing the gene-expression cassette...

  39. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    Published
    April 17, 2018, 1:37 p.m.
    ...piggyBac (PB) transposon: The PB transposon is a mobile genetic element that transposes via a “cut and...Generates transgene-free and vector-free cells Limited genomic integration Multiple transfections required...Delivery DNA-Free High High Transgene-free and vector-free Multiple transfections required Protein Delivery...vector, but transient expression of Cre recombinase allows for deletion of the inserted transgenes that are.... This system is transfected into cells as a donor plasmid containing the transposon, along with a helper...sequences, which the PB transposase recognizes. Following reprogramming, transgenes can be removed by re-...also includes measles and mumps. Sendai transduces a wide range of cell types and replicates in the cytoplasm...

  40. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    Published
    March 21, 2017, 2:30 p.m.
    ...forming membrane pores) transfection. These techniques are still used and efficiently transfer plasmid...and stable expression of the transgene   Transfer plasmids The transfer plasmid is the vector you clone...comes to transferring DNA into primary cells or non-dividing cells. To target these un-transfectable cells...Dox), the Tet-controlled transactivator (tTA, also expressed by your transfer vector) binds to the TRE...production and transduction Once you have cloned your gene of interest into lentiviral transfer vector, the...will first need to transfect producing cells, usually 293T cells, with your transfer plasmid and your packaging...you don’t have any experience in transfection, CaCl2 transfection is a simple and cheap technique that...

  41. Technical Design of a Western Blot

    Type
    Blog Post
    Published
    Aug. 22, 2024, 1:15 p.m.
    ...covers a size range appropriate for the target(s) and transfer method. Membranes The two membrane options ...) steps to the transfer: you’ll need to pre-wet most PVDF membranes before the transfer and allow them...Types of transfers Your transfer options are wet, semi-dry, or dry/electroblot. A wet transfer is the most... while semi-dry transfers require a semi-dry transfer cell. Dry/electroblot transfers only work in the...low-background PVDF membrane, it also has higher background noise than nitrocellulose membranes. Pro tip! You...visualize proteins on a PVDF membrane without staining; simply wet your membrane with 20% methanol and place..., meaning that it will transfer the most protein from your gel to your membrane. It requires anywhere ...

  42. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ...variant of PSmOrange, a photoswitchable fluorescent protein which transitions from emitting orange (λ= 565nm...enhancement of transcriptional activation via binding of multiple copies of a transcription factor to a ...the stop codon. Thus a non-translated mRNA will appear yellow, but a translated mRNA will be red. This system... were untranslated in the nucleus, confirming the hypothesis that most mRNAs are not translated until ...P-bodies are translationally repressed whereas the nonsequestered pool goes on to initiate translation. These...tool can also be used to visualize translocations, genome rearrangements and cancer-associated chromosome... been used to study transport characteristics of various Golgi and plasma membrane reporters as well as...

  43. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    Published
    March 8, 2021, 2:41 p.m.
    ...systems efficiently transfer a range of protein sizes in minutes without any transfer buffer. These systems...proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using antibodies. ...mixture of protein is separated in a gel, transferred to a membrane, stained with antibodies, and finally ...a broad size range to be separated on a single gel.  Immobilizing proteins on a membrane Once separated...migrate out of the gel and attach to the membrane.  In a wet transfer system the sandwich is completely submerged...filled with a transfer buffer formulated to conduct the electric current. The wet transfer process is very... very effective in transferring a broad range of protein sizes but takes hours to overnight.  In contrast...

  44. Plasmids 101: Terminators and PolyA signals

    Type
    Blog Post
    Published
    March 31, 2016, 2:30 p.m.
    ...initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination...from the transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically...generally serves to promote transcript longevity in eukaryotes and promote transcript degradation in prokaryotes...thought to destabilize the transcription complex, initiating cleavage of the transcript.   Figure 1: ...Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes  Delivered by ...terminators in the context of AAV transgene expression levels. Transcription Termination Rho-dependent termination...both prokaryotic and eukaryotic systems must be transcribed into RNA, which occurs in three phases: initiation...

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

    Type
    Blog Post
    Published
    March 17, 2016, 2:30 p.m.
    ...gene transcription (6). The EBOV genome contains seven viral genes, each flanked by a transcription start...replicated and transcribed in the cell when the appropriate viral proteins are provided in trans (see Other...genes and the minigenome are transcribed (and the viral mRNAs are translated) by cellular machinery. NP ...altering the amount transfected (see Technical Tips). Minigenome replication and transcription In order to replicate...it will terminate transcription and polyadenylate the mRNA. These mRNAs are translated by cellular host... lines that are highly transfectable.  The amounts of each plasmid transfected into cells are also extremely...interested in genome replication and transcription of negative strand RNA viruses.   References 1. Buchholz...

  46. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    Published
    Sept. 29, 2016, 2:30 p.m.
    ...plates. Step 2: Express random phage DNA fragments in E. coli Now that random DNA fragments have been ...nitrocellulose membranes and exposed to IPTG in order to induce protein expression. Cells on the membranes will ...orientation of each membrane on its plate before moving on. Then peel each membrane off the plate (saving...high salt.  To probe membranes with target bacterial cells, wash your membranes with PBST to remove salt... on the membrane to bind their target cells, pulling them out of solution. Wash your membranes well with...searches return no candidates. Our strategy: Express random fragments of the phage genome and probe with live...identified using this method. Step 1: Prepare a random fragment library of phage DNA This first step involves...

  47. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    Published
    Jan. 13, 2025, 9:49 p.m.
    ... non-edited strand, they can cause the cell to remake that strand using the edited strand as the template...the nicked target strand. The initial result is a heteroduplex, with overlapping strands of edited and unedited...Improving reverse transcriptase efficiency Building upon prior reverse transcriptase research, the Liu...the edit into one strand, there’s a mismatch between the original sequence on one strand and the edited ... the unedited strand away from the initial nick site (to avoid creating a double strand break), increasing...binds the edited strand, as in the PE3b system, guides nicking of the unedited strand only after editing...process and promote copying of the edited strand to the unedited strand (Chen et al., 2021). The team applied...

  48. Plasmids 101: Mammalian Vectors

    Type
    Blog Post
    Published
    March 25, 2014, 3:15 p.m.
    ... does not guarantee 100% transfection efficiency. Stable transfection A stable transfection is used to...process called transfection. Transfection is somewhat comparable to bacterial transformation (the introduction...the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection process...the genome to form a stable transfection. Unlike bacterial transformation, scientsts do not "select" for...fluorophore in a later post, so stay tuned! Transient transfection and the elusive "mammalian ORI" For many...plasmid depleted, but still want to use transient transfection methods. Since there are no "natural" mammalian...must be compatible with the cell type they are tranfected into – a bacterial origin of replication (ORI...

  49. Your Lentiviral Plasmid FAQs Answered

    Type
    Blog Post
    Published
    April 23, 2014, 1:08 p.m.
    ...considered. Q2: Can I use lentiviral transfer vectors for transient transfections? A2: Technically, yes. Will ...produced by co-transfecting three essential ingredients: the lentiviral packaging vector, a transfer vector including...specifically for transient expression (find some useful backbones here) or from viral transduction. While not...3rd-generation transfer vectors apart? A3: Addgene defines 2nd- or 3rd-generation transfer vectors based... Are transfer plasmids replication competent? What is SIN? A6: Most (if not all) Addgene transfer plasmids...enhancers and the transgene; such interactions can alter expression of not only the transgene but also that...which is rarely more than 30% of the transfer plasmid. Many transfer vectors are SIN. Packaging plasmid...

  50. Antibodies 101: Stripping and Reprobing Western Blots

    Type
    Blog Post
    Published
    Aug. 1, 2024, 1:15 p.m.
    ... your gel and membrane transfer as normal. Immediately after transfer, dry the membrane to maximize protein...you from having to run a whole new gel and membrane transfer. Let’s get started! Before you begin In the...manufacturer’s instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed ... as normal! Stripping your membrane Alright, so you’ve dried your membrane and returned it to buffer, ...’ll want to: Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...a fume hood. Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...you need to start a whole new blot? Never fear: membrane stripping is here! Rather than start over, you...
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