...strain. F- Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 λ- e14- mcrA0 relA1 rpsL150(strR) spoT1...that carried out the labor-intensive process of replicating your new plasmid: the Escherichia coli bacteria...conjugation. Genes listed on F´ are wild-type unless indicated otherwise fhuA (formerly tonA) ferric hydroxamate...vectors. Used by Addgene for pooled library amplification. F' mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1 gyrA96...typically standard; just having the gene listed indicates it is non-functional. If a gene is deleted, it... Most Popular Plasmids 101 Posts: Origin of Replication The Promoter Region – Let's Go! Mammalian Vectors...

...with fresh media containing selection reagent Days 3–14: Change media as needed Days 14–18: Stain cells...be killed and no colonies should be visible. Every 3–4 days, gently aspirate the media and replace it with...selective reagent for 12 days with media exchanges every 3–4 days. Colonies were then stained with 0.1% crystal...selective reagent for 12 days with media exchanges every 3–4 days. After 12 days of selection, no cells have ...without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) 0.1%...Figure 1: A549 cells were transduced with the indicated serial dilutions of the lentiviral vector pRosetta...Figure 2: A549 cells were transduced with the indicated serial dilutions of the lentiviral vector pHAGE...

... 1 µg DNA 1 µL of each Restriction Enzyme 3 µL 10x Buffer 3 µL 10x BSA (if recommended) x µL dH 2 O (to... T4 DNA Polymerase or Klenow DNA Polymerase I for 3′ overhang removal and 5′ overhang fill-in. If you ...The amount of DNA that you cut depends on your application. A diagnostic digest typically involves ∼500 ...volume usually varies from 10-50 µL depending on application and is largely determined by the volume of DNA...manufacturer’s instructions. Pro-Tip Depending on the application and the amount of DNA in the reaction, incubation...you will be using the digested DNA for another application (such as a digestion with another enzyme in a...70 °C for 15 mins, or purifying the DNA via a purification kit, such as a (Link opens in a new window) ...

...Cells Day 2–3 (am): Remove media, replace with fresh media containing selection reagent Day 3–14: Change...300 200 1:10 150 350 1:50 30 470 1:100 15 485 1:500 3 497 Add 0.5 mL of a single viral dilution to each ...cell death, the cell media should be changed every 2–3 days to maintain the dose of antibiotic, which may...without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) 0.45...lower dilutions depending on your downstream applications. If you’ve titered your virus beforehand, you...that the transgene has integrated in different locations in the various cells in the culture. This is because...

...end that is identical to an adjacent segment and a 3′ end that anneals to the target sequence. One strategy...reaction: T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back from the DNA 5’ end. Complementary...) to the isothermal reaction mix. ET SSB protects 3’ ssDNA ends from the ssDNA-specific endonuclease activity...product, gel-purify DNA segments. Otherwise, PCR purification or even the raw PCR mix can work fine in an ...

...separated by size, (2) transferred to a membrane, and  (3) detected using antibodies.   Figure 1: Overview...post-translational modifications, quantify protein levels relative to standards, detect the cellular location of proteins...pull-down assays.  Westerns are divided into two categories, native and denaturing. In a native western, ...matrix, the blotting membrane, the gel, a copper cathode/iblot gel matrix, and filter paper. Pretty much...search, first pay attention to the validated applications listed on the vendor’s website and choose an...western and vice versa.  In addition to validated applications, pay close attention to the controls listed ...and incubation times. If the vendor includes publications that cited the antibody, consider reviewing ...

...scientific enterprise and not just impact. Cause #3: Poor experimental design It’s easy to get excited...someone has replicated it precisely. You can find a great discussion of different types of replication in Nosek...perfect story for a “high impact” publication can lead to the publication of rare flukes showing positive...that fall into this category include performing experiments with too few replicates leading to suspect ...these positive results will be published. The publication record will then show that the tested hypothesis...reproducible publishing practices. One example is the publication of peer reviews so we can see how and why papers... also means that you’re more likely to get a publication (for better or worse, the currency for success...

...Originally published May 3, 2017 and last updated Sep 24, 2020 This post was contributed by guest blogger...the optimal location. It is also important to have good information on the exact location of the TSS. ...appreciable levels of modification. Whole-genome sequencing of cells modified by CRISPR indicates that the consequences...specific base edit, or modulation of gene expression. Location and sequence are important considerations for ... gRNAs. For indels, it's not so important what location in the gene you target, but it is important that...fewer sequences to choose from). Finally, for HDR, location is much more important because you have to target...target site is far more constrained by the desired location of edit; efficiency decreases dramatically when...

...spend the next 6 years. Know yourself I rotated in 3 labs before choosing a graduate school lab. All the...honestly evaluate your own style of work and communication before making this important decision. I’ll ...Prepare by going sleuthing on the lab website, publication records, LinkedIn, ResearchGate or Twitter. Be...the appropriate first authorship position on a publication of her project and made another lab member first...– including failure to acknowledge good work, allocation of meaningless tasks, removal of responsibility...connections such as serving on Boards? Does the publication record show evidence of industry collaboration...skills guide Learn about careers in science communication Read posts on mentoring for scientists Resources...

...image-based figures for scientific publications. PLOS Biology, 19(3), e3001161. https://doi.org/10.1371...protein is (localization) and how much there is (quantification). But what if you’ve got several interesting...primary antibodies that have been validated in IF applications (find IHC validated or ICC validated antibodies...Immunocytochemistry Protocols for other antibody applications...

... 20, 2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment Pipette controller...a 24-well cell culture treated plate. Seed 5 x 10 3 HeLa cells per well. Allow the HeLa cells to grow ... min in 500 µL PBS on a rocking platform. Section 3: Labeling with antibody Block for 20 min at RT on ...Guide Western Blot Protocol Recombinant Antibody Purification Protocol Introduction Immunocytochemistry is...as methanol or acetone may be better for some applications. Remove the paraformaldehyde and follow your...

...0: Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day 7 (am): Harvest cells Equipment...interest Triton X-100 Benzonase/DNAse I (Millipore 71205-3) 40% Polyethylene Glycol 8000 (PEG) + 0.5 M NaCl Cell... PBS and add 35 mL of 0.05% Trypsin/EDTA. Wait ~2-3 minutes for cells to detach. Gently tap the sides ...stir slowly at 4 °C for 1 h, then keep at 4 °C for 3 h without stirring to allow full precipitation. Precipitation... at 4 °C if needed. Transfer the entire sample to 3 x 500 mL conical bottles and centrifuge at 3900 rpm... of sonication to avoid overheating of the sample. Mix well between rounds of sonication. Sonicate until...container pH meter Stir plate Magnetic stir bar Sonicator Ear protection Vortex Reagents Adherent HEK293T...

...and can exhibit recombination levels as high as 1-3%. In light of this finding, we now include guidance...promoter, such as the truncated tyrosine hydroxylase (TH) promoter for catecholamine neurons or glial fibrillary... Flp), to either flip or excise a gene that is located between them. Early recombinase-dependent vector...expression profile at multiple steps from plasmid amplification in bacteria to transgene expression in vivo.... the level of spontaneous recombination. This indicates that the amount of leaky expression can largely...are typically employed during viral plasmid amplification (Fischer, KB. 2019). This means that all DIO...of spontaneous reversion during AAV plasmid amplification. On the left homologous pairs of scrambled cre-recombinase...

...Spinocerebellar ataxia 22 Bernard Weinstein 21238 PKC gamma CAT PRKCG HA CMV Spinocerebellar ataxia 21 Bernard Weinstein...TLS 2: hTLS.pCDNA1 FUS CMV ALS David Ron 21829 TLS 3: TLS.pRSET.C FUS His T7 ALS David Ron 21830 TLS 4:... APEX2 CMV Parkinson's Mark Cookson 165107 pGEX4T-3-6AOptParkin PRKN GST tac Parkinson's Arthur Haas 166671...dementia (FTD) Thomas Kukar 176922 pcDNA3.1(+)_Granulin 3+linker4 GRN Flag, Strep CMV Frontotemporal dementia...dementia (FTD) Thomas Kukar 176923 pcDNA3.1(+)_Granulin 3-no linker GRN Flag, Strep CMV Frontotemporal dementia... HA CMV ALS Catherine Tomasetto 104450 pEGFPC1-hVAP-B KD/MD VAPB GFP, HA CMV ALS Catherine Tomasetto 104465...

...about roles in this field, most jobs needed at least 3 months experience working in a developing tropical...at the Institute of Cancer Research in Sutton, located on the edge of London to gain relevant experience...recession. As each advertised job received many applications, employers were requesting that only scientists...commercial company. In my particular situation, communication between departments was sometimes difficult ... order to have meetings between teams. This communication is vital because otherwise I’d lose touch with...

...Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest antibody Equipment Biosafety...tube and vortex with three 1 s pulses. Incubate for 3 min at room temperature. Transfer the flask of HEK293...to mix. Return the flask to the incubator. Section 3: BCD Feed and valproic acid supplementation During...General Transfection Protocol Recombinant Antibody Purification Protocol Introduction Transfections allow for...antibody can be purified for use in a variety of applications. Sharing speeds science. We believe that sharing...antibody-expressing plasmids containing the SV40 origin of replication use a HEK293 line stably expressing the SV40 ... containing the Epstein-Barr virus origin of replication use a HEK293 line stable expressing Epstein Barr...

...anneal to the regions upstream (5’) and downstream (3’) of the DNA segment to be amplified. When these reagents... increase the time of the denaturing. Your 5’ and 3’ primers should be designed to have similar melting...Reverse Primers: Hybridize and are complementary to the 3’ ends of the sense and anti-sense strands of the template...adequately. Divalent cations such as Mg 2+ and Mn 2+ stabilize the buffer solution. These cations can also be ...used for PCR-mediated DNA mutagenesis. A higher cation concentration increases the error rate of the DNA... provide a suitable environment for the DNA amplification reaction. Reference Page Top Index...

...plasmid fingerprinting", where you cut the plasmid into 3-8 pieces such that all (or most) fragments are small...insert, but significantly off center (ideally around 1/3 of the way from one end), and also cuts in the backbone... . The pattern of the fragments on the gel can indicate if the plasmid contains the expected size insert...before use of more expensive forms of plasmid verification, such as DNA sequencing . In the example above...side of the insert, you can get a very obvious verification of the orientation so long as the expected products...

...would fail Addgene QC and would not be used. Section 3: Image Analysis Use ImageJ (Link opens in a new window...the curves for the protein bands as follows: Figure 3 Select Analyze . Select Gels . Select Plot Lanes ....Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 (HC) Peak 4 (LC) Total Area HC + LC Area Purity AR0018... Recombinant Antibodies Recombinant Antibody Purification Protocol Introduction This protocol describes...mobility of the bands (sample AR0016 in Figure 1) indicates that the samples may not have been processed correctly...ImageJ. Select File . Select Open . Choose the location of the file to open. Change the image type to ...select the Chart Type drop down menu and select Scatter chart. Change the data range and select the cells...

...Science 6.1 (2015): 50-69. Royal Society of Chemistry. 3. Ermolenko, Dmitri N., et al. "Observation of intersubunit...cellular impediments. FRET has been used in many applications, including drug discovery by big pharma (i.e... acceptor doesn’t need to go to the same exact location as the donor. As long as some of the acceptor ...C-terminus of your target protein, but even these modifications can affect the structure or function of certain... close to the donor, faulty delivery to other locations in the cell are not a problem, as long as they...The poly-histidine tag often used for protein purification is a reliable FRET acceptor labeling site when... way as NTA-sepharose beads used for protein purification: the NTA binds to the His tag on your protein...