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Addgene: Tetracycline Inducible Expression

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Link: https://www.addgene.org/collections/tetr

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Offer Details: The initial system Gossen and Bujard developed is known as tetracycline off: in the presence of tetracycline, expression from a tet-inducible promoter is reduced. To use tetracycline as a regulator of gene expression, a tetracycline-controlled transactivator (tTA) was developed. tTA was created by fusing tetR with the C-terminal domain of VP16 (virion protein 16), an essential transcriptional See more ...

Addgene: Optogenetics Guide

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Link: https://www.addgene.org/guides/optogenet

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Offer Details: Microbial opsins, such as those described below, can be targeted and expressed in specific subsets of neurons, allowing precise spatiotemporal control of these neurons by turning on and off the light source. Optogenetics has been broadly applied to study the physiology of the brain and nervous system to better map and understand neuronal circuits. Optogenetic tools have also been used to See more ...

Addgene: Cre-lox system

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Link: https://www.addgene.org/collections/cre-

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Offer Details: FLEx Switch ∗: This system allows scientists to utilize recombination elements such as Cre to turn off the expression of one gene while simultaneously turning on another. The FLEx switch system takes advantage of the orientation specificity of Cre and the different types of target sites available, both mutant and wild type. The ability to manipulate the number, orientation, and type of See more ...

Addgene: CRISPR Guide

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Link: https://www.addgene.org/guides/crispr/

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Offer Details: In addition to off-target activity, it is also important to consider factors that maximize cleavage of the desired target sequence or on-target activity. Two gRNA targeting sequences with 100% homology to their DNA targets may not result in equivalent cleavage efficiency. In fact, cleavage efficiency may increase or decrease depending upon the specific nucleotides within the selected target See more ...

Addgene: Plasmid Modification by Annealed Oligo Cloning

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Link: https://www.addgene.org/protocols/anneal

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Offer Details: Remove the hot block from the heat source (turn off or move block to bench top) allowing for slow cooling to room temperature (~45 minutes). Method 2. Place mixed oligos in a PCR tube. Place tube in a thermocycler programmed to start at 95°C for 2 minutes. Then, gradually cool to 25°C over 45 minutes. Ligation: Dilute 5μL of annealed oligos with 45μL nuclease-free water and quantify the See more ...

Addgene: Zhang Lab's CRISPR Frequently Asked Questions

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Link: https://www.addgene.org/crispr/zhang/faq

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Offer Details: The degree of off-target effects depends on a number of factors, including: how closely homologous the off-target sites are compared to the on-target site, the specific site sequence, and the concentration of Cas9 and guide RNA (gRNA). These considerations only matter if the PAM sequence is immediately adjacent to the nearly-homologous target sites. The mere presence of additional PAM See more ...

Addgene: pCSF107mT-GATEWAY-3'-LAP tag

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Link: https://www.addgene.org/67618/

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Offer Details: Mammalian Expression ; in vitro transcription or translation off of an SP6 promoter, Xenopus expression Promoter CMV and SP6 Tag / Fusion Protein. C-termnal LAP (eGFP-TEV-S) Growth in Bacteria. Bacterial Resistance(s) Chloramphenicol and Ampicillin Growth Temperature. 37°C See more ...

Addgene: Protocol - How to Run an Agarose Gel

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Link: https://www.addgene.org/protocols/gel-el

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Offer Details: Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box. (Optional) If you did not add EtBr to the gel and buffer, place the gel into a container filled with 100 mL of TAE running buffer and 5 μL of EtBr, place on a rocker for 20-30 mins, replace EtBr solution with water and destain for 5 mins. See more ...

Plasmids 101: Origin of Replication - Addgene

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Link: https://blog.addgene.org/plasmid-101-ori

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Offer Details: The origin of replication is the DNA sequence which allows initiation of replication within a plasmid by recruiting transcriptional machinery proteins, enabling a plasmid to reproduce itself. See more ...

Addgene: pAAV-hSyn Coff/Fon hChR2(H134R)-EYFP

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Link: https://www.addgene.org/55648/

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Offer Details: Plasmid pAAV-hSyn Coff/Fon hChR2(H134R)-EYFP from Dr. Karl Deisseroth's lab contains the insert ChR2(H134R)-EYFP and is published in Nat Methods. 2014 Jul;11(7):763-72. doi: 10.1038/nmeth.2996. Epub 2014 Jun 8. This plasmid is available through Addgene. See more ...

Plasmids 101: Restriction Cloning - Addgene

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Link: https://blog.addgene.org/plasmids-101-re

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Offer Details: You need to isolate your insert and backbone from the enzymes used to digest them as well as any pieces cut out or off of them. An easy way to do this is gel purification. In gel purification, you use a voltage difference across a gel matrix (usually agarose) to pull your negatively charged DNA through the gel. As indicated in the figure on the left, your digested DNA (and undigested controls See more ...

Addgene: LZF1827 pAAV_hSyn-Flp-off-Cre-on-stGtACR2-CFP

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Link: https://www.addgene.org/135413/

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Offer Details: Plasmid LZF1827 pAAV_hSyn-Flp-off-Cre-on-stGtACR2-CFP from Dr. Adam Cohen's lab contains the insert stGtACR2-CFP and is published in Cell. 2020 Jan 23. pii: S0092-8674(20)30047-7. doi: 10.1016/j.cell.2020.01.001. This plasmid is available through Addgene. See more ...

Addgene: Protocol - How to Create a Bacterial Glycerol Stock

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Link: https://www.addgene.org/protocols/create

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Offer Details: To recover bacteria from your glycerol stock, open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw! Streak the bacteria onto an LB agar plate. Grow your bacteria overnight at the appropriate temperature. Growth conditions, including copy number See more ...

Addgene: pLVX-TIGHT-CYCLINF-Tet-OFF

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Link: https://www.addgene.org/32977/

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Offer Details: Plasmid pLVX-TIGHT-CYCLINF-Tet-OFF from Dr. Michele Pagano's lab contains the insert CYCLIN F and is published in Unpublished This plasmid is available through Addgene. See more ...

Addgene: AAV Purification

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Link: https://www.addgene.org/protocols/aav-pu

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Offer Details: Use 1x PBS (or cell lysis buffer) to top off the tube. Seal the QuickSeal tubes. Centrifuge at 350,000 g for 90 min in a T70i rotor at 10 ℃. *Pro-Tip* If you need more time, you can alternatively centrifuge for 2 h at 200,000 g at 18 ℃. Carefully take the QuickSeal tubes out of the rotor and place them in a stable rack. **Make sure not to disturb the gradient!** Collect Fractions. Option See more ...

CRISPR History and Development for Genome - Addgene

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Link: https://www.addgene.org/crispr/history/

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Offer Details: Truncated gRNAs: Truncated gRNAs display less off-target activity. High fidelity Cas9s: Three high fidelity Cas9 variants, SpCas9-HF, eSpCas9, and HypaCas9 display very low off-target activity due to rationally designed mutations. Researchers have expanded the CRISPR field beyond SpCas9, moving us closer to being able to target every locus in every genome. Multiple Cas9 orthologs have also See more ...

Addgene: Cre-dependent OFF post-mGRASP

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Link: https://www.addgene.org/browse/article/7

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Offer Details: Jinhyun Kim Lab: Cre-dependent OFF post-mGRASP Unpublished. Plasmids from Article. ID Plasmid Purpose ; 51906: paavCAG-JxOFF-post-mGRASP-2A-dTomato: jxOFF-post: Loading Sign Up for Our Newsletter. Receive the latest news, hot plasmids, discounts and more. Sign Up. Subscribe to Our Blog . Learn about the latest plasmid technologies and research tools. Subscribe. Contact Addgene. Have See more ...

Addgene

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Link: https://www.addgene.org/search/education

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Offer Details: This website uses cookies to ensure you get the best experience. By continuing to use this site, you agree to the use of cookies. See more ...

Plasmids 101: The Promoter Region – Let's Go!

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Link: https://blog.addgene.org/plasmids-101-th

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Offer Details: Gets turned off with high levels of cellular tryptophan. lac: General expression: Promoter from lac operon: Constitutive in the absense of lac repressor (lacI or lacIq). Can be induced by IPTG or lactose. Leaky promoter with s omewhat weak expression. lacIq mutation increases expression of the repressor 10x, thus tightening regulation of lac promoter. Good for modulating gene expression See more ...

Addgene: Pooled Library Amplification

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Link: https://www.addgene.org/protocols/pooled

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Offer Details: *Pro-Tip* Scraped bacteria in LB can clog the pipette either mix gently up and down avoiding introducing bubbles or pour off plates into conical tubes as needed. Add 10 mL cold LB to each plate for each scrape and use spreader to scrape plates. *Pro-Tip* Pushing motion is better than pulling motion Take care not to split or gouge agar during the scraping process. Add each scrape into a 50 mL See more ...

Addgene: Pooled Libraries

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Link: https://www.addgene.org/pooled-library/

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Offer Details: Current research suggests that CRISPR screens may have fewer off-target effects than shRNA screens. Variants of CRISPR knockout screens include CRISPR activation (CRISPRa) and inhibition (CRISPRi) screens. These screens use catalytically dead dCas9 to inhibit or activate given genes without direct genomic modification. As in shRNA screens, the effects are reversible. When choosing your library See more ...

Addgene: Terms of Use

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Link: https://www.addgene.org/terms-of-use/

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Offer Details: Addgene retains the right to remove any Content or user-submitted information for any reason, including but not limited to, Content or user-submitted information that we deem threatening, demeaning, profane, obscene, a violation of intellectual property rights or privacy laws, off-topic, commercial or promotion of organizations or programs, or otherwise injurious or illegal. We also retain the See more ...

Addgene: Genome-scale measurement of off-target activity

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Link: https://www.addgene.org/browse/article/2

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Offer Details: Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Morgens DW, Wainberg M, Boyle EA, Ursu O, Araya CL, Tsui CK, Haney MS, Hess GT, Han K, Jeng EE, Li A, Snyder MP, Greenleaf WJ, Kundaje A, Bassik MC Nat Commun. 2017 May 5;8:15178. doi: 10.1038/ncomms15178. PubMed Article Plasmids from Article. ID Plasmid Purpose ; 101926: Bassik lab Human CRISPR See more ...

Addgene: Handling Plasmids from Addgene - Purifying

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Link: https://www.addgene.org/protocols/purify

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Offer Details: Pour off the supernatant, being careful not to disturb the bacterial pellet. Resuspend the pellet in 100 μl of cold Solution I. Vortex the solution for 2 mins or until all bacteria are fully resuspended. Add 200 μl of Solution II and invert the tube carefully 5 times to mix the contents. The contents will become clear and thicker as the proteins and DNA are denatured. *Pro-Tip* Do not vortex See more ...

Cpf1: A New Tool for CRISPR Genome Editing

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Link: https://blog.addgene.org/cpf1-a-new-tool

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Offer Details: Comparing Cpf1 and Cas9 on-target and off-target efficiency. When Cpf1 was first identified, we didn't know much about its on-target and off-target editing efficiency. Kim et al. and Kleinstiver et al. characterized genome-wide editing efficiency of two Cpf1 orthologs known to be active in mammalian cells, LbCpf1 and AsCpf1. In both reports, on target editing efficiency for the Cpf1 orthologs See more ...

Chemogenetics vs. Optogenetics: Which Method Should I Choose?

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Link: https://blog.addgene.org/chemogenetics-v

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Offer Details: The opsin ChETA, for example, is a ChR2 opsin mutated to have faster off kinetics and allow neurons to repolarize faster. Chemogenetics does not offer the same level of control over timing. Because chemogenetics relies on chemical activation and the diffusion of drugs throughout the body, the onset of receptor activation takes minutes. As a result, it is not possible to elicit an immediate See more ...

Plasmids 101: Inducible Promoters - Addgene

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Link: https://blog.addgene.org/plasmids-101-in

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Offer Details: In the OFF state, the promoter is inactive because a bound repressor protein actively prevents transcription. Once an inducer binds the repressor protein, the repressor protein is removed from the DNA. With the repressor protein absent, transcription is turned ON. Types of inducible promoters. Chemical agents, temperature, and light are all examples of factors that can lead to the induction of See more ...

Plasmids 101: Protein tags - Addgene

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Link: https://blog.addgene.org/plasmids-101-pr

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Offer Details: Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the accompanying cartoon, they have a multitude of uses including (but not limited to) purification, detection, solubilization, localization, or protease protection. See more ...

7 Simple Strategies to Resolve Conflicts with Difficult

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Link: https://blog.addgene.org/7-simple-strate

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Offer Details: Hands-off super busy types, who never have time to mentor you, and; Micromanagers, who question every detail of your project and work hours. While specific strategies to deal with each of these personality types is beyond the scope of this article, there are a few principles that will help you resolve most conflicts, even with difficult people. If I had to use one word for to summarize these See more ...

Plasmids 101: FLEx Vectors - Addgene

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Link: https://blog.addgene.org/plasmids-101-fl

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Offer Details: These Cre-off switches may be referred to as DO (D ouble-floxed O rientation) and could be used to study the physiology or behavior of certain cell populations. Acknowledgement: Through a partnership with genOway, we are able to distribute materials containing FLEx technology. Read the genOway press release for more information. References: 1. Schnütgen F, Doerflinger N, Calléja C, Wendling See more ...

Sequencing Options for CRISPR Genotyping

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Link: https://blog.addgene.org/sequencing-opti

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Offer Details: Off-target events due to >3 bp mismatches or that are sequence-independent are rare, but they are detectable using just genome-wide unbiased methods. However, most investigators use single cell clones for in vitro CRISPR experiments. The likelihood that a single cell clone derived from the pool contains both the rare off target event and the desired edit is low. Therefore, unbiased sequencing See more ...

Addgene: pAAV-hSyn-DIO {mCAR}off-{DTR-GFP}on-WPRE

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Link: https://www.addgene.org/111395/

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Offer Details: Plasmid pAAV-hSyn-DIO {mCAR}off-{DTR-GFP}on-WPRE from Dr. Adam Kepecs's lab contains the inserts mCAR and DTR-EGFP and is published in Neuron. 2018 Jun 6;98(5):905-917.e5. doi: 10.1016/j.neuron.2018.05.028. This plasmid is available through Addgene. Image: Illustrated plasmid map in PNG format. GenBank File: Plasmid sequence and annotations. Use text editor or plasmid mapping software to view See more ...

Enhancing CRISPR Targeting Specificity with eSpCas9

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Link: https://blog.addgene.org/enhancing-crisp

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Offer Details: The off-target problem. It is well known that CRISPR/Cas9 genome editing can result in unwanted changes at non-target sites. Means to decrease these so-called “off-target effects” have included using a Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA sequences used for targeting; however, these options can be cumbersome, can lower on-target efficiency, and sometimes even See more ...

Academic vs. Industry Postdocs - Addgene

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Link: https://blog.addgene.org/academic-vs.-in

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Offer Details: Also, while you’re unlikely to be laid off as an industry postdoc since your funding is usually separate from a project’s or a team’s, it is possible that your mentor (and even their department) could be laid off while you’re there. Of course, similarly, an academic advisor could also lose their funding. Communication: In both industry and academia, you will have to communicate your See more ...

A New Generation of Adenine Base Editors Improves Editing

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Link: https://blog.addgene.org/adenine-base-ed

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Offer Details: Reducing off-target editing in ABE8s. While base editors are precise molecular tools, they do have undesired off-target editing effects. ABEs have been described to introduce base changes in cellular DNA and RNA dependent or independent of the gRNA used (Gaudelli et al., 2017). The authors tested ABE8.17-m and found that one base change mutation (ABE8.17-m+V106W) (Gaudelli et al., 2020) was See more ...

How to Design Your gRNA for CRISPR Genome Editing

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Link: https://blog.addgene.org/how-to-design-y

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Offer Details: The off-target activity of gRNAs is important to consider. While the basic landscape of mismatches that can nevertheless still lead to activity has been established, and can be used to identify sites that are likely to give rise to an off-target effect, there’s not enough data to fully predict which sites will and will not show appreciable levels of modification. Whole-genome sequencing of See more ...

Addgene: pAAV-CBA-DO(FAS)-GCaMP6s

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Link: https://www.addgene.org/110135/

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Offer Details: Cre-off expression of GCaMP6s Depositing Lab. Bernardo Sabatini. Publication. Markowitz et al Cell. 2018 May 10. pii: S0092-8674(18)30512-9. doi: 10.1016/j.cell.2018.04.019. ( How to cite ) Sequence Information. Sequences (2) See more ...

CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems

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Link: https://blog.addgene.org/anti-crisprs-sw

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Offer Details: Reducing off-target effects. Prolonged Cas activity can increase the chance of off-target editing particularly after the majority of on-target editing has occurred. Anti-CRISPR proteins can be used to limit this off-target editing, but when is the best time to shut off Cas activity? See more ...

Interview: Ed Boyden on Optogenetics, Neuroscience, and

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Link: https://blog.addgene.org/mits-ed-boyden-

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Offer Details: Boyden: Optogenetics is a toolset that lets you turn the electrical activity of neurons on or off using pulses of light. By activating a neuron or set of neurons, you can see how it contributes to behavior or pathology, and by disabling neurons you can figure out what they are necessary for. See more ...

Choosing a CRISPR Nuclease: Site Accessibility

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Link: https://blog.addgene.org/choosing-a-cris

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Offer Details: As these off-target effects can be genome-wide and, in some cases, difficult to determine, the potential for off-target cleavage should be an important consideration during any experiment. Sensitivity. Cas9 sensitivity is often closely related to specificity. Sensitivity is a measure of the on-target activity of the Cas9 nuclease. This can be determined by measuring the indel frequency at a See more ...

CRISPR 101: Ribonucleoprotein (RNP) delivery

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Link: https://blog.addgene.org/crispr-101-ribo

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Offer Details: Off-target effects can occur due to prolonged Cas9 expression; The requirement for Cas9 transcription and translation delays editing; Cas9-gRNA ribonucleoproteins. One alternative approach, which avoids many of these complications, is to directly deliver a ribonucleoprotein (RNP), consisting of the Cas9 protein in complex with a targeting gRNA, to your cells of interest. There are many See more ...

A Guide to Designing a Scientific Poster: Content, Layout

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Link: https://blog.addgene.org/guide-to-design

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Offer Details: But, before you can show off your work at a conference, you need to first make your poster. While a poster generally contains the same sections as a primary research article, it’s important to understand that presenting your work in poster format differs in many ways from writing a manuscript. If you have ever attended a scientific conference, or even a lab recruiting session at your See more ...

CRISPR Meets Synthetic Biology: A Conversation with MIT’s

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Link: https://blog.addgene.org/crispr-meets-sy

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Offer Details: But there are a couple of problems. You couldn’t do it without repeating the same guide RNA twice. The second problem was the shape of the response function or how the output changes as a function of input. Usually that function is in the shape of an S; there is a narrow threshold like a switch that turns on or off. CRISPR shows a more See more ...

Controlling for Off-target Effects with a New Genome-wide

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Offer Details: Off-target cutting does not confound results of CRISPR screen. Next, the toxicity of guides with off-target sites was profiled. Guides with exact or 1-bp mismatch off-targets had greater toxicity than guides that had zero mismatch off-targets. Guides with 2-bp mismatch off-targets were only toxic if they had 5+ off-target sites. Results from the screen also reproduced several previously See more ...

Multiplexed Capture of Promoter-enhancer 3D Chromatin

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Link: https://blog.addgene.org/capture-ing-chr

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Offer Details: They found that DNA sequences targeted by the gRNAs were the top enriched sequences in the experiment and there was little enrichment of DNA targeted by a negative control gRNA or predicted gRNA off-target sites. Long-range chromatin interactions identified with the multiplexed CAPTURE 2.0 largely replicated interactions previously identified with the original CAPTURE method. Both CAPTURE See more ...

Addgene: Optimized sgRNA design to maximize activity and

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Link: https://www.addgene.org/browse/article/1

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Offer Details: Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE Nat Biotechnol. 2016 Jan 18. doi: 10.1038/nbt.3437. PubMed Article Plasmids from Article. ID Plasmid Purpose ; 73178: Human sgRNA library Brunello in lentiGuide-Puro See more ...

Lighting up

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Link: https://www.ruecouponcode.com

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Offer Details: humble jellyfish Aequorea victoria, which floats in waters off the U.S. Pacific coast. It was largely ignored until the latter half of the 20th century, when a scientist became curious about its beautiful bioluminescent properties and found it possessed an amazing molecule: green fluorescent protein (GFP), which exhibits bright green fluorescence when exposed to blue or violet light See more ...

The Scientific Conference Poster Session: Tips for Success

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Link: https://blog.addgene.org/scientific-conf

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Offer Details: Remember your outline: Instead of getting bogged down in the minutia or coming off as a broken record, remember the outline you created for yourself. Hit the key bullet points and elaborate if your audience seems captivated. Expertly navigate questions. Answering questions is arguably the most difficult part of a poster session, but with a little preparation and a few deep breaths the day-of See more ...

How Dry Ice Affects Viral Vector Sample pH & How to Avoid It

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Link: https://blog.addgene.org/how-dry-ice-co2

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Offer Details: We’re always looking for ways to improve our shipment processes. After reading a publication describing how short term storage on dry ice can shift sample pH, we wondered whether or not the dry ice we use to keep viruses frozen during shipment was having an impact on the samples.We therefore devised a few experiments to determine if our tubes were permeable to the CO 2 released from dry ice See more ...

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