...organism where deletion strains are available. Then, staff or students clone similar sequences from a less-...

...away from a desired site does not have the same devastating impact on efficiency as 10-15 bps away does. ...

...techniques do tip the scales in your favor. For starters, Golden Gate cloning is extremely efficient due...

...useful tools, and as such, we’re always looking to understand our community better. We therefore looked at ...

...translate perceived signals to physical actions. Understanding neuronal pathways and the function of certain...

...where cleavage leads to the loss of spectinomycin resistance. xCas9 3.7 showed the broadest PAM range, recognizing...

...and adaptations that can provide an increased understanding of DNA binding and gene regulation. For additional...

...not a leader,” because you don’t see yourself  standing in front of a crowd and using your charisma to...

...extremely important gene in animal development. To understand where sonic hedgehog got its name, we need to...

...medical journal. Decades later and after years of painstaking work trying to isolate the causative gene for...

...IgG isotype standard 2.5 mg/mL - can use commercial standard or validate an in-house standard Before Starting...Thaw IgG standard and prestained protein ladder on ice. Reagent Preparation Prepare IgG Standard Dilute ...ready-to-use bio-safe Coomassie G-250 stain (Addgene uses SimplyBlue SafeStain) and ImageJ software. The sample... serial dilutions of a standard antibody of known concentration. After staining with Coomassie blue, protein...ready-to-use biosafe Coomassie stain and ImageJ software. Protocols... Protocols Coomassie Purity Stain of Recombinant Antibodies...Antibodies Coomassie Purity Stain of Recombinant Antibodies You may also like... Antibody Plasmid Collection...

... Protocols Generating Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus... antibiotic resistance (e.g., puromycin, blasticidin), which enables selection of a stable cell culture...lentiviral vectors deliver antibiotic resistance. This protocol was established using 293T cells but can be adapted... of antibiotic, which may not be stable at 37 °C. To achieve a stable cell pool, the antibiotic selection...expression. As polyclonal populations of resistant cells start proliferating and the individual wells become...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...Introduction This protocol can be used to generate stable cell lines expressing a gene of interest from an...

...help stabilize the standards add carrier DNA to a final concentration of 4 ug/mL to each standard dilution... Once a validated standard curve is obtained, make a small aliquot of each standard (enough for 1 or 2...their initial value. If the Ct value of the standard starts to drift, it’s time to make a new one. When...your plasmid standard with DNase ** Make 6 serial dilutions, in duplicate, of your standard curve plasmid...5'-CGGCCTCAGTGAGCGA ITR-containing plasmid for standard curve RNase-free DNase 10X DNase buffer Nuclease-free...Pipette tips General Considerations Always run standards and samples in duplicate at least If possible,... the samples have been added to the qPCR plate. Start by adding water, then SYBR master mix, then the ...

... carry one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the...to be resistant to the antibiotic. On the second plate, streak out a strain that’s not resistant to the... be resistant to the antibiotic, (+) indicates that the tested strain is supposed to be resistant to the...Negative Result 3: Only the Non-resistant Strain Grows If only the non-resistant strain grows, it’s likely ... carrying them. The presence of an antibiotic resistance gene on a plasmid allows researchers to easily...for the selection of only those bacteria with resistance to that antibiotic - usually conferred by a plasmid... plasmid carrying the antibiotic resistance gene. The following protocol will allow you to make your own...

...transfer stack. Set the Top Stack to one side and discard the white separator. Keep the Bottom Stack in the...SDS-PAGE running buffer Prestained protein ladder Ethanol iBlot 2 PVDF Mini Stack, Thermo Fisher IB24002... of the BSA standard that range from 0–2000 µg/mL . In duplicate, dilute 10 µL of standard, blank, and...blank from all of the samples. Plot a standard curve of BSA standard concentration versus absorbance. Extrapolate...antibody Secondary antibody Deionized water Before Starting Refer to the manufacturer's instructions for additional...total protein concentration of the sample from the standard curve. Determine the volume of sample required...the samples into the gel. Load 5–10 µL of the prestained protein ladder. Place the lid on the tank and...

...markers. Note this assay requires staining resistant colonies with crystal violet solution and, therefore...Change media as needed Days 14–18: Stain cells and count resistant colonies Equipment Class II, Type A2...Filter 0.1% crystal violet solution through a 0.22 μm filter to remove any precipitates. Stain each well...well with 1 mL of 0.1% crystal violet for 10 min at RT. Gently remove the stain. Wash cells 3x with 1 mL...exchanges every 3–4 days. Colonies were then stained with 0.1% crystal violet and counted. Figure 2: A549 cells...lentiviral preparations that confer antibiotic resistance. Protocols...lentivirus preparations that confer antibiotic resistance. Determining the titer of your lentiviral preparation...

...protocol begins with a stable cell pool. For details, see our protocol for generating stable cell lines with ...Store at 4 °C. Polyclonal stable cell pool: see our protocol for generating stable cell lines with lentivirus... monoclonal cell line from a polyclonal pool of stable cells. Protocols... monoclonal cell line from a polyclonal pool of stable cells. Transducing cells with lentivirus results...cell populations that are more likely to retain stable transgene expression. Other methods of generating...Hemocytometer or other cell counter Reagents Polyclonal stable cell pool DMEM high glucose, Corning 10-013-CV ...CV L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated...

...power objective is directly over the stage. It is best practice to start with the lowest power objective to...microscope stage. If using a slide, you can secure it into place using the metal clips on the stage. Turn ...Turn on the power source and use the stage arm to move the stage so that the light shines onto your sample...Once your sample is in focus, use the stage arm to move the stage (and ultimately your slide) until your... out more protocols and videos to help you get started in the lab! Introduction Microscopes are emblematic....com. Base Light Source Condenser and Diaphragm Stage Objective Lenses Focus Knobs (Fine and Coarse) Nosepiece...of light hitting the sample or b) using dyes or stains that add color to some components of the sample...

...introduced, the puromycin resistance marker encoded in pLKO.1 allows for convenient stable selection. Figure ...with effective gene silencing: Starting at 25nt downstream of the start codon (ATG), search for 21nt sequences...homologous recombination of unstable regions, such as Invitrogen Stbl3™ or NEB Stable cells. This will ensure...Stock solution is stable at 4°C for up to one year. The powder form of polybrene is stable at 4°C for several...drives expression of puromycin. Puro R Puromycin resistance gene for selection of pLKO.1 plasmid in mammalian...bacterial origin of replication. Amp R Ampicillin resistance gene for selection of pLKO.1 plasmid in bacterial...should be 36-52%. Sense 3’ end should have low stability – at least one A or T between position 15-19. ...

...Plasmid DNA DNA Ligation Background Information A standard Polymerase Chain Reaction (PCR) is an in vitro...became automated with the discovery of a heat resistant DNA polymerase from the thermophilic bacterium... Thermus aquaticus (Taq) . Taq polymerase can withstand many heating and cooling cycles, which would denature... template strand to the point where the strands start denaturing and the hydrogen bonds are broken between...seconds at 55°C: The forward and reverse primers are stable within this temperature range to anneal to each...DNA template strands. The DNA polymerase is also stable enough to now bind to the primer DNA sequence. ...Taq Buffer with MgCl 2 : Provides an optimal and stable chemical environment for the DNA polymerase to ...