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We narrowed to 451 results for: abo.5

Showing: 401 - 420 of 451 results

  1. CRISPR 101: Any Base Transversion Editors

    Type
    Blog Post
    ...), 4897. https://doi.org/10.1038/s41467-024-49343-5 Tong, H., Wang, X., Liu, Y., Liu, N., Li, Y., Luo,...In our last post, we talked about the first base transversion editors: CGBEs, or C → G Base Editors. ...apyrimidinic sites and abbreviated as AP sites. A collaborative team from the David Liu and Dali Li labs reported... CRISPR 101: Cytosine Transversion Editors Read about four base editor reporters Resources on Addgene.org...

  2. New Optogenetic Tools for Cytoskeleton and Membrane Control

    Type
    Blog Post
    ...left-right organizer development. PLoS Genetics, 19(5), e1010765. https://doi.org/10.1371/journal.pgen.1010765... Check out our Optogenetics Guide to learn more about how these modules work, and read on to learn how...to the cell membrane isn’t powerful enough, what about altering the chemical composition of the membrane...

  3. Plasmids 101: Expressing shRNAs for RNA Interference

    Type
    Blog Post
    ...–297. https://doi.org/10.1016/s0092-8674(04)00045-5 De Rie, D., Abugessaisa, I., Alam, T., Arner, E., ... & Corey, D. R. (2021). Reexamining assumptions about miRNA-guided gene silencing. Nucleic Acids Research...

  4. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ... concentration: 4 × 10 5 cells/mL To seed one 96-well plate, make 10 mL of a 5 cell/mL solution. Calculate...conditioned medium prepared above to make a new cell solution at a concentration of 5 cells/mL. Prepare approximately...high glucose, add 55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Polyclonal stable...0.45 µm PES filter or centrifugation at >500 x g for 5 minutes. Do not use the medium if the cells are overly...this cell solution in fresh complete medium. This 5 cells/mL solution will be used to seed the 96-well...the total cells needed: Total cells needed: 10 mL × 5 cells/mL = 50 cells Determine the volume of homogenized...homogenized cell solution needed: (50 cells)/(4 × 10 5 cells/mL) = 0.125 µL Because this is such a small ...

  5. AAV ddPCR Titration

    Type
    Protocol
    ...targeting ITR: ITR Forward Primer: 5’-CGGCCTCAGTGAGCGA ITR Reverse Primer: 5’-GGAACCCCTAGTGATGGAGTT ITR Probe...pipette to add 5 µL of each viral sample to Dilution 1 in the 48-well dilution plate and pipette 5–10 times ...time. Dilution 1 (20X): 5 µL in 95 µL 1X PCR buffer (1:20) Dilution 2 (20X): 5 µL in 95 µL 1X PCR buffer...400) Dilution 3 (20X): 5 µL in 95 µL 1X PCR buffer (1:8,000) Dilution 4 (20X): 5 µL in 95 µL 1X PCR buffer...buffer (1:160,000) Dilution 5 (20X): 5 µL in 95 µL 1X PCR buffer (1:3,200,000) Dilution 6 (2X): 50 µL in...dilution series. For dilutions 1–5, use the 1–10 µL multichannel pipette set to 5 µL. For dilutions 6–8, use...PCR tubes. Add 5 µL of dilutions 6–8 to the appropriate PCR tubes. Pipette back and forth 5 times. Lightly...

  6. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...oligo) primers, which anneal to the regions upstream (5’) and downstream (3’) of the DNA segment to be amplified...Repeat steps 2-4 25-30 times. Final Extension for 5 minutes at 72°C: A final extension to fill-in any ... tube Ice Bucket 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 mM...36.8 μL Sterile dH 2 O 0.2 μL Taq DNA Polymerase (5 units/μL) PCR Machine Agarose Gel Procedure Primer...reagents on ice): 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 mM...Reverse Primer (10 μM stock) 0.2 μL Taq DNA Polymerase (5 units/μL) 36.8 μL Sterile dH 2 O (variable) Note: ...reaction tubes in PCR machine. Set annealing temperature 5°C below the primer melting temperature (Tm). Set extension...

  7. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...genomic DNA. Incubate tube on ice for 5 min. Centrifuge the tube for 5 min at 12,000 g. Notes: Pellet contains...Optional: Add 5 μL of 2 mg/mL RNase A to the supernatant in the new tube and incubate at 37 °C for 5 min. Note...are worried about losing the pellet. Dry with vacuum or by inverting over paper towel for 5-20 min. Resuspend...200 μL of Solution II and invert the tube carefully 5 times to mix the contents. The contents will become...purified plasmid DNA. Incubate solution on ice for 5 min. Add 150 μL of cold Solution III to each tube....microfuge tube for 30-60 sec. Centrifuge the tube for 5 min at room temperature on the highest setting. Note... overnight OR -80 °C for 30 min OR on dry ice for 5 min. Note: This freezing may help the DNA to precipitate...

  8. Scientific Reproducibility - Focusing on Solutions at the Minisymposium on Reproducibility

    Type
    Blog Post
    ...stories and read papers wherein a researcher tries 5 separate experiments at once to get confirmatory data...easier to find information about research tools. BenchSci collates information about antibodies while FPbase...variety of additional resources and information about organizations promoting reproducibility in this ...that make it easier to share in-depth information about reagents, protocols, analysis tools, and more. Being...from a freezer. As you’ll see in Steve Almo’s talk about work at the Institute for Protein Innovation (IPI... talked to many researchers who have complaints about the unreliability of antibodies. IPI is working ...FPbase curates information about fluorescent proteins so you can best apply these technologies to your own...

  9. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    ...Nature. 2015 Dec 3. doi:10.1038/nature.2015.18947 5. Siva K, Covello G, Denti MA. Exon-skipping antisense...estimated that very little dystrophin correction (about 4%) is needed to see muscle improvement, with only...diseases we’d like to treat with CRISPR. As seen above, DMD can be treated with “one-size-fits-most” NHEJ-mediated.... Precision genome editing faces the challenges above and more. One chief challenge is upregulating HDR...

  10. Antibodies 101: Validation

    Type
    Blog Post
    ...Validation (Uhlen, et al., 2016). This group proposed 5 Pillars of antibody validation which represent five...may not be the most accessible option for many laboratories. Additionally, it can be difficult to distinguish...the captured proteins off for mass spec analysis (above). When the results come back it shows that the majority...our Selecting an Antibody blog post! Learn more about Antibodies and their many applications in our Antibodies...

  11. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    ...–203 . https://doi.org/10.1016/0003-2697(81)90281-5 Pillai-Kastoori L, Heaton S, Shiflett SD, Roberts ... the thickness of the band provides information about the relative abundance of the protein in the sample...history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using...

  12. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    ...inducing site-specific DNA methylation. Biology Open 5:866–874 . https://doi.org/10.1242/bio.019067 Find...modifications. Like the TALE-LSD1 system described above, dCas9-LSD1 inactivates targeted enhancers. Although...setting up your experimental system. Let us know about your experience with these constructs in the comments...

  13. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...restriction site (GAATTC) to the 5’ end of this primer, making our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC...consist of: Leader Sequence: Extra base pairs on the 5' end of the primer assist with restriction enzyme ...Therefore, our Forward Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds..., resulting in a final Forward Primer sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse...final 18bases of the ORF, including the stop codon (5'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC...restriction enzyme digestion. This gives us a sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with...put the sequence we chose for our reverse primer (5’-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this...

  14. CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

    Type
    Blog Post
    ...773–779 . https://doi.org/10.1038/s41477-020-0704-5 Santana I, Wu H, Hu P, Giraldo JP (2020) Targeted ... Addgene. In addition to the plasmids described above, Addgene carries several useful CRISPR toolkits ...

  15. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins. Optional...buffer to each of your DNA samples and mix well. Use 5 µl of loading buffer per 25 µl of sample. Note: Loading...container filled with 100 mL of TAE running buffer and 5 μL of EtBr, place on a rocker for 20-30 mins, replace...replace EtBr solution with water and destain for 5 mins. Using any device that has UV light, visualize your...electrophoresis (PAGE), which is typically used to separate 5 - 500 bp fragments. How do you get better separation...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr... of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample...

  16. Protocol - How to Design Primers

    Type
    Protocol
    ...guanine or cytosine is used at the 3’ end, and the 5’ end of the primer usually has stretches of several...) of 50-60°C Primer pairs should have a Tm within 5°C of each other Primer pairs should not have complementary...If you will be including a restriction site at the 5’ end of your primer, note that a 3-6 base pair "clamp...amplification process. When designing, if unsure about what nucleotide to put at a certain position within...capabilities. Taking into consideration the information above, primers should generally have the following properties...

  17. Affinity Purification of Recombinant Antibodies with Protein A or Protein G

    Type
    Protocol
    ...mL conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm...pipette Reagents Aspirating pipette, VWR 414004-265 5 mL pipette, VWR 89130-896 10 mL pipette, VWR 89130...PD10 Buffer Reservoir, Millipre Sigma GE18-3216-03 5% Sodium Azide, 500 mL, VWR 7144.8-16 PBS, 1X pH 7.4...tubes. Cap the Gravitrap columns at the bottom. Add 5 mL of Pierce IgG Elution Buffer to the capped Gravitrap...hydrochloride pH 9.0. Cap the tubes and vortex for 5 s to mix. Determine the protein concentration of each...Discard the flow through into a waste container. Add 5 mL PBS to the column. Centrifuge at 1000 x g for 2...personal protective equipment including laboratory coat, laboratory goggles, and gloves. Sodium azide containing...

  18. A Guide to Getting Started in Undergrad Research

    Type
    Blog Post
    ...a lab is nothing like a typical part-time or 9-to-5 job. I had to ask to find out what a “postdoc” is,...dig deep Talk to your professors about their research, and learn about what makes them excited to do what...sciences), people conduct research to answer questions about how the world works. As scientists, we probe beyond...curious person. I like to ask questions and think about the best way to answer those questions. I’ve met...enjoy, is worthwhile and can help us make decisions about what we want to do in the future. How do I get started...sense of what kind of research you would be excited about. I know quite a few friends who opted to join labs... a lot out there worth exploring! Being excited about the science allows you to ask more interesting questions...

  19. Lentivirus ddPCR Titration

    Type
    Protocol
    ...viral stock 240 µL 400 µL 5 200 µL of 2.5-fold dilution 200 µL 400 µL 10 200 µL of 5-fold dilution 200 µL ...heat inactivated premium grade, fetal bovine serum 5 mL glutaGRO 50 U/mL benzonase: 15 mL DMEM Complete...before seeding. Mix each well with a 1 mL pipette 5–10 times. The final volume in the well is 1.5 mL, ...transfer to a microcentrifuge tube. Centrifuge for 5 min at 100 x g . Gently aspirate supernatant. Wash...Wash cell pellets in 500 µL PBS. Centrifuge for 5 min at 100 x g . Gently aspirate supernatant. Extract ...additional 10-fold dilution into 6-well plate 2.5 25 5 50 10 100 20 200 40 400 80 800 160 1600 To calculate...0.2045454545 8.18E+06 4 100 3180 20540 0.3096397274 6.19E+06 5 50 8960 17080 1.049180328 1.05E+07 6 25 14440 24260...

  20. Plasmids 101: Using Transposons in the Lab

    Type
    Blog Post
    ...piggyBac, which is a big advantage over viral vectors (~5 kB payload for AAV and ~8 kB payload for lentivirus...Addgene blog Learn about the INTEGRATE system for bacterial genome engineering  Read about the Sleeping Beauty...DNA transposons are most commonly used in the laboratory for genome manipulation. When transposons are...Applications of transposons Now that you’ve learned about some of the popular transposon systems, let’s take...2020) Jump around: transposons in and out of the laboratory. F1000Res 9:135. https://doi.org/10.12688/f1000research...Beauty DNA transposon Learn about the piggyBac DNA transposon ...
Showing: 401 - 420 of 451 results