... Thaw 4 tubes of electrocompetent cells on ice for 15-20 minutes or until completely thawed. Chill a box...obtained. Ensure that electrocompetent cells are being used. Chemically competent cells will not provide... to the inherent possibility of altering the composition of the library. Bottlenecks, fitness differences... in the morning to ensure that outgrowth and competition are limited. Equipment Table top centrifuge BioRad...MicroPulser TM , Bio-Rad 1652100) Reagents 200 µL electrocompetent cells (Default: 4 tubes of Endura Duos, Lucigen...Stbl4 cells or other ultra-high efficiency electrocompetent cells that are suitable for unstable or recombination-prone...recombination-prone DNA. The use of electrocompetent cells is essential to ensure high efficiency uptake...

...layout is complete, select 'Run' to begin the droplet reading. When the droplet reading is complete, export...biosafety level 2+. Please ensure that you are in compliance with your institution’s biosafety regulations...Human, Bio-Rad, 10031244 Reagent Preparation DMEM Complete: 500 mL DMEM, high glucose, pyruvate 55 mL heat...serum 5 mL glutaGRO 50 U/mL benzonase: 15 mL DMEM Complete 3 µL of 250 U/µL benzonase Procedure Transducing...the following dilution series of virus in DMEM complete: Prepare 7 microcentrifuge tubes with the appropriate...Leave one well untransduced (add 150 µL of DMEM complete instead of a viral dilution). Remember, this is...Gently add 1.5 mL of 50 U/mL Benzonase in DMEM complete to each well. Incubate at 37 °C for 30 min. Gently...

...want to use electro-competent cells instead of the more common chemically-competent cells. The number of...sufficient to give specificity and to also be compatible with a standard PCR reaction. Therefore, our...design is similar, but we need to use the reverse complement to get PCR amplification. We can start similarly...the ORF). We now need to generate the reverse-complement of this sequence so that we can successfully ...amplify the ORF. You can generate the reverse-complement using existing software (a quick internet search... one restriction enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you...according to the manufacturer’s instructions for your competent cells. For most standard cloning, you can transform...

...fragment of interest) into a compatibly digested vector backbone. This is accomplished by covalently connecting...are compatible, meaning that the overhanging base pairs on the vector and insert are complementary, the...is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells... sticky ends on either side of the vector are compatible with each other, the vector is much more likely... will allow you to verify that the vector was completely digested and if phosphatase treated, that the...Interpretation Uncut vector - Checks viability of competent cells and verifies the antibiotic resistance of... 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in the ballpark...

...Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...colony formation assay. Prepare a batch of DMEM complete containing 10 μg/mL polybrene by diluting 20 μL...fold serial dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene: Dilution Volume...Lentivirus or Previous Dilution (μL) Volume of DMEM Complete Containing 10 μg/mL Polybrene (μL) Volume of Virus...follows: Dilute 7,000 cells into 9.45 mL of DMEM complete containing 10 μg/mL polybrene. Mix well by pipetting...to 8 h and the media can be replaced with DMEM complete. However, antibiotic-containing media should not...aspirate the media from the cells. Add 1.5 mL of DMEM complete containing the appropriate antibiotic. Incubate...

...of a compound light microscope with the main components labeled: Figure 1: Diagram of a compound light...electron microscopes that allow us to see cellular components with science-fiction levels of detail. You can...common types that you're likely to encounter: the compound light microscope. As the name suggests, light ... make it through the sample because different components of the specimen will refract and reflect the ...) using dyes or stains that add color to some components of the sample but not others. The light that ...that allow you to capture images directly to a computer, but many microscopes do not and you may have ...

...want to use electro-competent cells instead of the more common chemically-competent cells. The number of...version of your insert flanked by restriction sites compatible with the recipient plasmid's MCS. However, you... one restriction enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you...Follow the manufacturer’s instructions for your competent cells. For most standard cloning, you can transform...transform 1-2μl of your ligation reaction into competent cells such as DH5alpha or TOP10. If using much...getting colonies, you might want to use higher competency cells. Additionally, if your final product is...you used only one enzyme or used enzymes with compatible overhangs you will need to verify the orientation...

...puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...kills all of the cells. Prepare a batch of DMEM complete + 10 µg/mL polybrene by diluting 20 µL of 10 mg...a range of dilutions of the lentivirus in DMEM complete + 10 µg/mL polybrene. Note, this is just a sample...Dilution Volume of Lentivirus (μL) Volume of DMEM complete + 10 µg/mL polybrene (µL) 0 0 500 1:5 300 200 ...,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or...all the media in these wells was made with DMEM complete + 10 µg/mL polybrene, the final concentration ...aspirate the media from the cells. Add 1.5 mL of DMEM complete containing the appropriate antibiotic. This is...

... mM NaCl, 2 mM MgCl2) Reagent Preparation DMEM Complete : 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...min. Neutralize trypsin by adding 10 mL of DMEM Complete. Pipet back and forth vigorously multiple times...-175 flasks. Adjust volume to 300 mL with DMEM complete media and mix. Seed all cells in 1 CS2. Return...CS2 to help detach the cells, add 200 mL of DMEM complete media, then transfer the cells into a sterile ...sterile bottle. Rinse the CS2 with 100 mL of DMEM complete medium and pool with the cells harvested in the previous... with PEI and allow for the addition of other components, such as sorbitol and sodium bicarbonate, sorbitol...Pipet back and forth to resuspend each pellet completely. Combine resuspended pellets and keep on ice....

... 21-mer targets in your gene B.2 Order oligos compatible with pLKO.1 C. Cloning shRNA oligos into pLKO...vector. A.2 Map of pLKO.1 pLKO.1 is a replication-incompetent lentiviral vector chosen by the TRC for expression...sequence), while the EcoRI site is preserved. For a complete map of pLKO.1 containing the 1.9kb stuffer, visit... nuclear import of the vector’s preintegration complex in the transduced cells. hPGK Human phosphoglycerate...about off-target effects. B.2 Ordering Oligos Compatible with pLKO.1 To generate oligos for cloning into... shRNA sequence flanked by sequences that are compatible with the sticky ends of EcoRI and AgeI. Forward...M0202S T4 DNA ligase buffer NEB #B0202S DH5 alpha competent cells Invitrogen #18258-012 Qiaquick gel extraction...

... 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Friday: Plate...your insert of interest Reagent Preparation DMEM Complete (also known as D10): 10% v/v FBS and 4 mM L-alanyl-L-glutamine...Plate 8×10 5 cells in a T75 flask in 15 mL DMEM Complete. Do not add pen-strep to the media. Use cells ...packaging cells at 3.8×10 6 cells per plate in DMEM Complete in 10 cm tissue culture plates. Incubate the cells...at RT. During the incubation, add 10 mL of DMEM Complete to a 15 mL conical and an appropriate amount of...the media. Replace the media with 10 mL of DMEM Complete or OptiPro SFM. Incubate the cells. Virus can ...

...works for both of your enzymes. Most companies will have a compatibility chart, such as the (Link opens in...enzymes that have compatible sticky ends. See (Link opens in a new window) NEB's compatible cohesive ends...ends chart . If you cannot find compatible sticky ends, you will need to fill in the overhangs and conduct...Master Mix" consisting of all of the reaction components except for the DNA. Aliquot your DNA into individual...

...strand completely; it is essential, however, that the 3’ end of the primer corresponds completely to the...reaction. One needs to design primers that are complementary to the template region of DNA. They are synthesized...sequences on the template molecule (must be complementary to template strand). However, primers do not... of each other Primer pairs should not have complementary regions Note: If you will be including a restriction...

...CAATTG ). The bottom oligo will be the reverse compliment so that they can anneal. Top oligo: 5' - CATATG...bp We also need to include additional bases to complement the overhangs generated when digesting the vector...could leave off the 3’ G on each oligo (and the complementary C of the other oligo), but this would destroy...following oligos from your favorite oligo synthesis company: Top oligo: 5' - AATTCCATATGTTAATTAAGGCGCGCCCAATTGG...annealed oligos). Transform 2-3μL into your favorite competent bacteria and plate. Be sure to pick multiple colonies...

...biosafety level 2+. Please ensure that you are in compliance with your institution’s biosafety regulations...Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...follows: Dilute 525,000 cells into 14 mL of DMEM complete. Mix well by pipetting or inverting. Aliquot 2... Prepare dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene. Note, this protocol...Volume of Lentivirus Stock (μL) Volume of DMEM complete (μL) Volume of 10mg/mL polybrene (μL) 1:10 150...

...DMEM complete containing 25 µM chloroquine diphosphate and incubate ~5 h For 10 mL of DMEM complete, add...Syringes for filtering Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...HEK293T cells at 3.8x10 6 cells per plate in DMEM complete in 10 cm tissue culture plates. Incubate the cells...the media. Replace the media with 15 mL of DMEM complete. Incubate the cells 24–48 h before checking for...

...Refrigerator Pipette controller Benchtop centrifuge compatible with 50 mL conical tubes NanoDrop spectrophotometer... 250 mL sterile bottle, VWR 430281 0.45 µm PES complete filtration unit, 500 mL, rapid-flow, VWR 73520...Place a mark on the side of the column where the compacted resin is slanted upward. Place column in centrifuge...centrifugation step Pro-Tip Resin will appear compacted after centrifugation. Discard the flow through.... Slowly apply the sample to the center of the compact resin bed. Pro-Tip For sample volumes <1.5 mL top...

...denaturation, annealing, and synthesis. After the PCR is complete, the product can be verified based on size by ...Diluting Primers Most people order primers from a company, which synthesizes and ships them as a lyophilized...essential that the template DNA and primer are complementary for a PCR to work efficiently. Taq Buffer with...Forward and Reverse Primers: Hybridize and are complementary to the 3’ ends of the sense and anti-sense strands...

...Corning 430167 (optional) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...Each 10 cm dish should be seeded in 10 mL DMEM complete, which will generate enough conditioned medium...conditioned medium, prepare this cell solution in fresh complete medium. This 5 cells/mL solution will be used ...adding 10 µL homogenized cell solution to 990 µL complete medium. Instead of transferring 0.125 µL, now ...

...layout is complete, select 'Run' to begin the droplet reading. When the droplet reading is complete, export... on the insert. Please ensure that you are in compliance with your institution’s biosafety regulations...Stabilization 98 10 2 1 Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to the Droplet Reader. Open...

...restriction digest protocol ). After the digest is complete, you will need to separate the linearized vector... A typical T4 Pol reaction is shown. Mix the components (add the T4 last) and incubate at room temperature...of T4 Pol in the following step. This may be accomplished by gel purification . Step 5: Create Insert ...

... and using a centrifuge to separate different components in a liquid sample....laboratory equipment used to separate different components in a liquid sample. It achieves this by using... you use, most will have essentially the same components. The photo to the left shows a front view of ...

...2009). Regardless of fragment length or end compatibility, multiple overlapping DNA fragments can be joined...overhangs by chewing back from the DNA 5’ end. Complementary DNA fragments can subsequently anneal to each... DNA Ligase - covalently joins the annealed complementary DNA fragments, removing any nicks and creating...

...bacterial strain and you will not need to obtain more competent cells and retransform. Bacteria on an LB agar ...streaking onto LB agar will prevent it from thawing completely and will improve the shelf life. It is very important...

...protocols are the building blocks for many more complicated procedures. Plasmid Cloning Protocols for constructing... and using a centrifuge to separate different components in a liquid sample Using a Light Microscope Learn...

...biosafety level 2. Please ensure that you are in compliance with your institution’s biosafety regulations...tips Pipettor and pipettes Benchtop centrifuge compatible with 50 mL conical tubes Automated cell counter...

...vivo . The iodixanol gradient in this protocol is composed of steps that separate out contaminants from an...exchange and concentration Note: Both steps could be completed in one (long) day. Equipment Ultracentrifuge T70i...

...). Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, ...room temperature for 20-30 mins, until it has completely solidified. Pro-Tip If you are in a hurry, the...

...bacterial cultures , please see those pages. Several companies, such as (Link opens in a new window) NEB , (Link...but within a smaller volume of buffer that is compatible with the next solution. Add a denaturing solution...

...science. General Considerations The ELISA can be completed in a single day or broken up over several days...ELISA plate washer. Equipment Spectrophotometer compatible with 96-well plates 1–10 µL single channel pipette...

...to allow for dissociation of the nucleoprotein complexes. The effectiveness of your RNA isolation will ...

...plasmid. It shows you each component, with the ID of the component plasmid, component name, size, and any comments...module, are the components. Using the handy website, users can simply select the components they want for...able to select components that work with your design. Note that you can browse all components by clicking...Fragmid doesn’t have the components I want? You can make your own Fragmid components! Ensure your backbone... Each module of Fragmid represents a class of components, such as your CRISPR enzyme or Pol 2 promoter...’ terminus, and selection markers. Need fewer components? A spacer can be inserted instead, allowing everything...everything to stay in the correct order. Most Fragmid components are available for request on the Addgene website...

...easily design, generate, and test constructs composed of complex genetic elements. To demonstrate the usefulness...several GMAP-compatible vectors for a wide variety of biological applications. First, GMAP-compatible backbones...mammalian systems. The DULIP system is comprised of Gateway-compatible plasmids in which “bait” proteins are...plasmids designed to complement unused auxotrophies in Saccharomyces strains by compensating for histidine (... do so have been hindered by a lack of tools, compounds, and assays to monitor activation of these receptors...including a stop codon) formats. The collection is compatible with both standard Gateway cloning and the FNLCR...assembly platform for generating both simple and complex DNA constructs Have you ever wished you could ...

...DNA strands complementary and non-complementary to the gRNA, respectively. When complexed with a truncated... (nuclease-competent) Cas9 interacting with either a full-length gRNA (20nt complementarity to target ...) or truncated gRNA (≤15nt complementarity to target site). When complexed with a full-length gRNA, native...with which it is complexed rather than the protein itself. When native Cas9 is complexed with a gRNA that...transcriptional activator to the Cas9-gRNA complex. When this complex is directed to a target upstream of the...Figure 3: Gene repression with nuclease-competent Cas9. When complexed with a truncated gRNA, Cas9 is still... directed to virtually any genetic locus when complexed with an appropriately designed small RNA, or guide...

...replication components are called helper plasmids. Below, we’ll breakdown the vector components first by ...nucleocapsid viral components needed for viral packaging, assembly, and display. Pol, the last component, contains...there are bonus components to these vectors that aren’t reflected in the common components outlined above...lentiviral guide outlines these components.     Fig. 1 - Viral vector components of retrovirus  Adeno-associated...production reasons, multiple plasmids with many components are used to package lab grown viruses. It can... protocols! In reality, there are many shared components across viruses (AAV, lentivirus, etc.) and their... what gag and pol stand for, we demystify all components of viral vectors here! Need Virus? Check out...

...role at a commercial virology testing company. This particular company carries out clinical trials of drugs...treatments for external companies. It was really exciting to work in a larger commercial company; the pay was good...the company and that my ideas and changes were welcomed, which is a benefit over larger companies which...obtaining work in scientific research would be very competitive, and so I applied for a summer role as a Research...This was particularly interesting as I had just completed my thesis on the study of e2Fb, which is a tumour...without issues. Because the primary purpose of the company was to fulfill contracts, my work sometimes felt...strong point. Working in industry: virology On completing my MSc, I again had to look at what type of work...

...different components, but all contain a FRET pair, a genetic component, and a small molecule component. Early...developed voltage sensor with a genetic component. The VSD components have historically come from a variety...have been engineered to have kinetics comparable to its one component counterpart and is significantly brighter...cellular localization component is typically handled by either the fluorescent component (i.e., when the dye...tools have two main elements: a voltage-sensing component and a fluorescent output. The GEVI family of gadgets... a hybrid approach and utilize both a genetic component as well as a small molecule which we will describe...reporter and the mechanism of their voltage sensing component, which separates them into two distinct categories...

... as Cascade (CRISPR-associated complex for antiviral defense) complexes, while Class 2 use single protein...effector complex after binding to a target. Class 1 is the most abundant of the two, comprising about 90%...tools using a Cascade complex is tricky, as you need to ensure every unit of the complex expresses at similar...have similar compositions and mechanisms, with the biggest differences being the components present in ...in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with a guide RNA...primary effector complex but are defined by the accessory Cas proteins in the Cascade complex. Despite a tenuous...the Cas effector may have the same name but be completely different! They are merely linked by a similar...

...are compatible with all your components. Next, consider the best form for those CRISPR components based...introducing those CRISPR components into your target cells. Delivery of these components into mammalian cell...most effectively achieved with different CRISPR components. Select the appropriate CRISPR tools for your...will you be editing? Do you want to deliver the components as DNA, or would mRNA or protein delivery be ...of the first steps is to identify what CRISPR components you will need to deliver. Minimally, one or more... the best method for introducing those CRISPR components into your target cells.  Mammalian CRISPR expression...best practices for efficient delivery of CRISPR components. If you are new to CRISPR or your model system...

...information about the cell’s complexity. A cell with a high degree of internal complexity such as extensive membranes... prepare sets of controls to aid with compensation. Compensation is the process of correcting spectral...need to analyze a specific subset of cells in a complex population. Have you spent hours in the biosafety...cell’s size, granularity, and target protein composition. It is used for a variety of applications such...and detecting specific populations of cells in a complex tissue or sample (1). When combined with cell sorters...instrument collects information about the cell’s size, complexity and fluorescence intensity.   What flow cytometers... cytometers detect Cell size and complexity, measured using visible light scatter The optical system has...