...at 95 °C, a common lysis temperature, so consider a longer incubation (10–20 minutes) at 70 °C, or a truly... Additionally, protein lysis should be done at 4 °C or on ice, whichever is more practical. If your lysing... truly lengthy incubation (30–60 minutes) at 37 °C, if you suspect aggregation in your prep. If your proteins...protein-based blockers degrade fairly quickly, even at 4 °C, so when in doubt, make up a fresh batch.  Antibodies...and time. Though the most common conditions are 4 °C overnight or room temperature for 1–2 hours, there...sample loaded Incubate primary antibody at 4 °C overnight Incomplete washing Ensuring wash is... may need to sonicate your lysates in order to release the proteins from their binding. If you’re seeing...

...interactions New auxin inducible degron system for C. elegans New CRISPR plasmids New items from the viral...tool-kit for the auxin inducible degron (AID) system in C. elegans By Alyssa Cecchetelli The auxin inducible... degron (AID) system has been extensively used in C. elegans since 2015 to conditionally degrade proteins... from Ashley et al., 2021.   The AID system relies on a transport inhibitor response 1 (TIR1) expressed...induce DSBs within minutes. New CRISPR plasmids for Borrelia burgdorferi from Christine Jacobs-Wagner’s lab...

...guest bloggers Roodolph (Roo) P. St Pierre and Rose C. St Pierre, founders of STEAMid. Internships open ...guest bloggers Roodolph (Roo) P. St Pierre and Rose C. St Pierre! Roodolph (Roo) P. St Pierre is currently...populations. Roo is the co-founder of STEAMid.       Rose C. St Pierre is currently a graduate student at Northeastern...certain to choose your recommenders based on your relationships to the person as well as their current sphere...

... one-copy ‘housekeeping’ gene), and quantified relative to a 'normal' sample with known copy number set...nice thing about qPCR is that since it measures relative quantification - that is, since you’re looking...looking at the amount of DNA in your sample relative to your normal control - you can set up your calculations...provides absolute quantitative data, instead of relative quantification.  Even though no one option is ... M., Sim, B., Mustafa, M., Nik Yusof, N. K., Lee, C. K., Abu Bakar, S., AbuBakar, S., Hollox, E. J., &...

...and T7E1 have been widely adopted due to their relative simplicity and low cost. The problem with these... detection rates mean the investigator can be relatively certain that their samples don’t have off-target...PubMed Central PMCID: PMC4349094. 3. Bell, Charles C., et al. "A high-throughput screening strategy for... 22963480. PubMed Central. 5. Tsiatis, Athanasios C., et al. "Comparison of Sanger sequencing, pyrosequencing...genotyping, is critical to demonstrating causal relationships between genotype and assayed phenotype. Verifying... for validating edits.  Mismatch cleavage assays rely on pairing between the edited strand and wild-type...limit of detection of ~5%. This means they do not reliably detect editing events that occur in less that ...

... promoter, gRNA and pol III terminator. These relatively large cassettes (considering the gRNA itself ... vector. In addition, the pol III promoter is relatively weak, and low expression of gRNAs from these ...specific genome editing. Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ...

...Williams, Eric J. Ross, Sofia M. C. Robb, Sarah A. Elliott, Kimberly C. Tu, Alejandro Sánchez Alvarado,... first discovered and characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a...gene expression and functions of key regeneration-related genes in four inexpensive and commercially available...

...requested! Fire Lab C. Elegans Kit - Our first popular "kit," Andrew Fire's set of vectors for C. elegans research...infographic created by our own Addgenie, Laura Veckerelli. Click on the picture below to see the full graphic...

...conformations: (A) bent-closed, (B) extended-closed, and (C) extended-open. When a ligand binds, it typically ... with much lower affinity than their open states (C). Figure courtesy of IPI. IPI anti-integrin toolset...and resources References Hao, Y., Yan, J., Fraser, C., Jiang, A., Anuganti, M., Zhang, R., Lloyd, K., Jardine...Jaumouillé, V., Hao, Y., Coppola, J., Yan, J., Waterman, C. M., Springer, T. A., & Ha, T. (2022). Single-molecule...courses on technologies and laboratory techniques related to antibody discovery. You can find cell surface...

...GFP (sfGFP) and mNeonGFP can fold in <10min at 37°C, mCherry takes ~15min, TagRFP ~100min and DsRed ~10hours...instance, enhanced GFP (EGFP) was optimized for 37°C, and is therefore most suited for mammalian or bacteria...GFPS65T is better suited for yeast studies (24-30°C). Brightness: Brightness is a measure of how bright...For a breakdown of GFP, it's variants, and their relevant mutations, check out Marcy's post from last week...not narrow enough to distinguish between closely related colors. Furthermore, most FPs  have a broad range...College of Medicine. He is interested in everything related to gene expression, particularly at the RNA level...

...organisms and cell lines. Although ChIP-seq is a relatively simple and robust protocol it does have limitations...digestion, fragments are released from nuclei by a short incubation at 37 °C. These short DNA fragments...or more transient DNA-protein interactions, the reliance on fixation does increase the chance of false-..., which stands for ‘Cleavage Under Targets and Release Using Nuclease,’ was recently developed by the ...strong detergent to permeabilize cells rather than to rely on nuclei extraction. They report that this change...

...four reprogramming factors- OCT4, SOX2, KLF4, and C-MYC - that induce a differentiated somatic cell to...additional pluripotency stage) Less Safety Less (C-MYC, a known oncogene is one of the reprogramming ...22645348. PubMed Central PMCID: PMC3390875. 25. Patsch, C., et al., Generation of vascular endothelial and smooth... PubMed Central PMCID: PMC4363833. 35. Eguizabal, C., et al., Dedifferentiation, transdifferentiation,...1): p. 82-94. PubMed PMID: 23329641. 36. Jopling, C., S. Boue, and J.C. Izpisua Belmonte, Dedifferentiation...2011. 12(2): p. 79-89. PubMed PMID: 21252997. 37. Merrell, A.J. and B.Z. Stanger, Adult cell plasticity in...2016. 17(7): p. 413-25. PubMed PMID: 26979497. 38. Thorel, F., et al., Conversion of adult pancreatic alpha-cells...

... Cas9 into n-terminal (Cas9(N)-2xNLS) and c-terminal (Cas9(C)-2xNLS) fragments facilitates target DNA ...activation, the c-terminal cas9 fragment was fused with FK 506 binding protein 12 (Cas9(C)-FKBP-2xNLS) and...out there (and particularly CRISPR), it is now relatively easy to design constructs and repair templates...an n-terminal DNA recognition domain and a mostly c-terminal nuclease domain. Feng Zhang's group utilized...Treatment with rapamycin induces Cas9(N)-FRB-NES and Cas9(C)-FKBP-2xNLS dimerization and net influx into the nucleus...genes using dCas9-VP64 activator fragments (dCas9(C)-FKBP-2xNLS-VP64 and dCas9(N)-FRB-NES). This system...constructing fusion proteins with customizable N- and C-terminal modules   Thanks to the lab of Tomás Santalucía...

...   References and Resources   References Beck C, Zhang D, Gong Y. Enhanced genetically encoded voltage...: 10.1016/j.cobme.2019.10.010. Bando, Y., Grimm, C., Cornejo, V.H. et al. Genetic voltage indicators.... 71 (2019). doi:10.1186/s12915-019-0682-0. Beck, C., Gong, Y. A high-speed, bright, red fluorescent voltage...heartbeat – a muscle contraction – is directly related to the electrical impulse of the cardiac cells....and this voltage is particularly physiologically relevant in neurons, myocytes, and endocrine cells. It ...

...science.aas9129   Zhang W, Yin J, Zhang-Ding Z, Xin C, Liu M, Wang Y, Ai C, Hu J. In-depth assessment of the PAM compatibility... Cas9 NG - SpG Cas9  NG  - SpRY nuclease  C to T Base Editor SpRY A to G Base Editor SpRY   ... NYN PAMs, but with less efficiency and less reliability. Notably, the SpRY variant didn’t display higher...

...difficult to grow. Growth Temp Choose from 30 °C, 37 °C, or room temperature. Growth Strain Please...frog), D. rerio (zebrafish), D. melanogaster (fly), C. elegans (nematode), S. cerevisiae (budding yeast)...be indicated in the “Species Other” column. Relevant Mutations Indicate the amino acid change as ...

...For example, entering ‘C. elegans’, or ‘elegans’ will filter the table to show C. elegans gRNA sequences...the time that would be spent making and testing entirely new gRNA designs. You can now easily find many...

...The modularity of antibodies means that it is relatively easy to physically divide up the different regions...fragments intended for use as primary antibodies are relatively uncommon. Though, if you want to use Fab fragments...features labeled. V = Variable domain. L = Light chain. C = Constant domain. H = Heavy chain. Created with biorender.com...

...scatter (FSC) and provides information about the relative size of the cell; larger cells produce more FSC...experiment, you would need a way to determine the relative contribution of yellow and orange from FITC and...interest with a fluorescent reporter on the N- or C-terminus of the protein. One downside of fusion proteins...doi.org/10.1002/cpim.40 Picot J, Guerin CL, Le Van Kim C, Boulanger CM (2012) Flow cytometry: retrospective...intensity of events. Since intensity positively correlates with expression, the further the shift of a cell...

...high Cas9 MOI considering that A549 cells are relatively easy to transduce. I ended up with too many Cas9...on genomic DNA. The surveyor assay itself was relatively easy. Figure 4: Detection of edited BRAF... new scientific observations. References 1. Rago C, Vogelstein B, Bunz F. Genetic knockouts and knockins...Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE. Nat ...