...as the protocol remains the same.    Figure 2: Microscopy-based antibody experiments should include...times with the same samples and compare the results across runs. If the results vary considerably, then the...protein level of a target that is thought to be equal across all samples, such as a housekeeping protein (GAPDH...tubulin). A loading control confirms equal loading across samples and equal protein transfer from the gel...

...this year. Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor plasmid contains...components of CRISPR/Cas9 (Cas9 protein and the crRNA-tracrRNA complex) and the donor plasmid into the cells ...

...out for help so you can get the support you need. 2) Prepare an agenda for every meeting Supervisors are...will need to be more assertive to get your point across. Some supervisors might try to intimidate you with...

...communicate the nuances of protein structure in a flat 2-D images. In the early days of structural biology,...large ribosomal subunit, he kept sliding his palm across the surface of the protein where the nascent polypeptide...

...follows: Figure 2 Using the box tool, draw a box around the entire first gel lane (as in Figure 2). Select Analyze... Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 (HC)...choose Use Equation . Select the Show R 2 checkbox. Pro-Tip The R 2 of the trendline should be between 0.95...Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... Add 5 µL of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to each sample. Spin the sample...bottom part of the gel where dye is visible. Section 2: Staining the Gel Place the gel in a plastic tray .... Figure 1 Recombinant antibody preps should have 2 clear bands at ~50 kDa and ~25 kDa corresponding to...

... iRFP713, % Oligomeric state Photo-stabilityc, t1/2, s pKa Brightness in HeLa cells vs. iRFP713, % d ...Fluorescent Proteins: Multiplexing and Optogenetics across Scales. Trends Biotechnol 36, 1230-1243 (2018)....near-infrared fluorescent proteins for multicolor imaging across scales. Nat Commun 11, 239 (2020). Oliinyk, O.S...

... 95 10 2 1 Denaturation 95 0.5 2 50 Annealing/Extension 60 1 2 50 Signal Stabilization 98 10 2 1 Hold ...should decrease by a factor of 2 across the dilutions. In the example below, 2-fold serial dilutions of a ... considered biosafety level 1 but may require BSL-2 handling depending on the insert. Please ensure that...single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette 20–200 µL multichannel ...20X): 5 µL in 95 µL 1X PCR buffer (1:20) Dilution 2 (20X): 5 µL in 95 µL 1X PCR buffer (1:400) Dilution... DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel pipet, load 20 µL of the reaction...Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to the Droplet Reader. Open the QuantaSoft software...

...) 12 (>100) xCas9(3.7)-ABE 69 (1.4) 43 (2) 21 (3) Not examined 16 (>100) Average percent...off-target activity. Surprisingly, off-target effects across 5 gRNAs with NGG PAM sequences were actually lower...

...PMID: 25538079. PubMed Central PMCID: PMC4353587. 2. Stephen, Andrew G., et al. "Dragging ras back in ... in the kit, the transcript most commonly found across all TCGA tumor samples is represented, making the...

... replication competent virus [usually HIV-1]; and 2) the potential for oncogenesis through insertional...screening with CRISPR and Lentivirus Read about the ClonTracr Library Resources on Addgene.org Addgene's most...

...temperature between 50-60°C for your PCR primers. Step 2: Linearize Vector In this example, the vector is cut...20-30 Eluted DNA 10-50 ng/μl 1 dGTP (100mM) 2.5 mM 2 DTT (100 mM) 5 mM 1 BSA (10 μg/μl) 0.25 μg/μl 1 T4...your treated vector and insert at a molar ratio of 1:2 or 1:3, using between 20 and 50 ng of vector per annealing... reaction is now ready for transformation. Use 1-2 μl of annealing reaction for each transformation, ...reactions by holding the vector concentration fixed across multiple concentrations of insert, thereby increasing...

...that numbers are not switched around or misread.  2. Track the container - It is just as important to ...weeks in transit and some have been transported across oceans and continents. Each deposit may consist...

...experience and to build upon your existing skills.  2. Find your niche Identify and develop an area of expertise...passion  Skills such as research, collaboration across multiple disciplines, writing, project management...

...externally fertilized, transparent progeny (Figure 2). These features give it a leg up (or a fin up?) over...1,500 zebrafish mutants that are still used in labs across the globe (Nüsslein-Volhard et al., 2012). One ...

...replicate, then purify the DNA.     Figure 2: A pooled library page with protocols boxed in red...centrifuging. Spinfection transduction efficiency can vary across cell types and generally requires optimization ...

...agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2% gel). Mix agarose powder with...gels are commonly used in concentrations of 0.7% to 2% depending on the size of bands needed to be separated...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds...allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases...for visualizing very short DNA fragments is polyacrylamide gel electrophoresis (PAGE), which is typically...

...backbone, which expresses the gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene...backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors then used GMAP assembly...engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand...number of tandemly expressed gRNAs respectively) and 2) the size of the deletion. Smaller deletions generally...portable method for bacterial genome engineering across multiple species Recent discoveries in the fields...

...Day 1: Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day ...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...channel pipette 200–1000 µL single channel pipette CO 2 incubator Pipet controller Hazardous waste container...approximately 50–60% confluent. For 293T cells this is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish should...the highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal ...inferred by the differences in colony size. Figure 2: Cas9 expression in monoclonal cell lines generated... events and the site(s) of proviral integration across cells. Selective pressure on this heterogeneous...

...s) Buffer BSA (if recommended by manufacturer) dH 2 O up to total volume Pro-Tips The amount of DNA that... 10x Buffer 3 µL 10x BSA (if recommended) x µL dH 2 O (to bring total volume to 30µL) The amount of restriction...from 45 mins to overnight. For diagnostic digests, 1-2 hours is often sufficient. For digests with >1 µg ... This will save you time and ensure consistency across the reactions....

...positive cells (Martin et al., 2019). Figure 2: Editing efficiencies using base editing reporters... sorting using FACS which could limit their use across different cell types and systems. To introduce ...