...need to determine these transduction patterns empirically for their own experiments. To this end, we offer...

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... this limitation, one option is to perform a hierarchical assembly, assembling fragments in multiple steps...

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...use the 2A peptides instead of an IRES when stoichiometrically equivalent levels of expression are required...

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...generally segregate with the cell pellet during purification procedures. Even if you produce a large amount...

...below. The GUS gene (β-glucuronidase) with histochemical GUS-staining is a very useful visual reporter...

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...Vector by Gel Purification Run your digest DNA on an agarose gel and conduct a gel purification to isolate...analyzing the gene’s function. The process is shown graphically in the following cartoon, in which we are adding...amplify and design primers that will bind to and replicate it. The following image shows the ends of the ...need to use the reverse complement to get PCR amplification. We can start similarly, taking the final 18bases...the (Link opens in a new window) QIAquick PCR Purification Kit . The PCR product is now ready for restriction...plasmid. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting...reaction and 1μg of recipient plasmid. It is also critical that as much of the recipient plasmid as possible...

...10x magnification. To determine the final magnification of your image, multiply the magnification of your...lab! Introduction Microscopes are emblematic of biological research and are found in many different types...suggests, light microscopes take advantage of the physical properties of light to detect small objects. Two...most important properties of a microscope are magnification (the ability to make an image larger) and resolution...reaches your eyes. These lenses determine the magnification of the image and the resolution your microscope...information, but the most important (for now) is the magnification power, such as 4x, 10x, or 20x. A 4x objective...times, and so on. The ocular lens also provides magnification and the power should be provided on the microscope...

...page you'll find practical lab protocols that you can use for a wide range of applications, with videos for...Video! DNA Purification Miniprep, phenol-chloroform extract, and precipitate DNA DNA Quantification Measure...protocols are the building blocks for many more complicated procedures. Plasmid Cloning Protocols for constructing...preparations. Antibodies Protocols for common antibody applications. Intro to the Lab Bench Name Description (Link...bacterial strain Watch the Video! CRISPR Library Amplification Amplify CRISPR pooled-plasmid libraries Diagnostic...Cloning by PCR Generate restriction sites by PCR Modification by Annealed Oligo Cloning Add a short stretch...Generating Stable Cell Lines with Lentivirus Genomically integrate your DNA sequence into a cell line ...

...Load 5 μL of each standard in duplicate Load 5 μL of each sample in duplicate. Do not forget to include a...dilution is appropriate). Quality of duplicates: Exclude duplicates from analyses if there is more than...Nuclease-free water Microcentrifuge tubes 96-well optical plate Pipette tips General Considerations Always...Always run standards and samples in duplicate at least If possible, include an AAV reference sample of known...should also contain an internal passive reference (typically ROX dye), to normalize non-PCR–related fluorescence...vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical: do NOT treat your plasmid standard with DNase...DNase ** Make 6 serial dilutions, in duplicate, of your standard curve plasmid (2 x 10 9 stock made in step...

...vector by gel purification Run your digested DNA on an agarose gel and conduct a gel purification to isolate...treatment of your recipient plasmid as well as a specifically designed test digest later to verify that the...plasmids. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting...plasmid and 1μg of recipient plasmid. It is also critical that as much of the recipient plasmid as possible...prior to the ligation step or prior to the gel purification step, depending on the phosphatase you choose...isolate the DNA. When running a gel for purification purposes it is important to have nice crisp bands and...bands away from the gel via your favorite gel purification method, it is important to determine the concentration...

... of sonication to avoid overheating of the sample. Mix well between rounds of sonication. Sonicate until...am): Harvest cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...container pH meter Stir plate Magnetic stir bar Sonicator Ear protection Vortex Reagents Adherent HEK293T...pyruvate, Corning 10-014-CV (optional) 7.5% sodium bicarbonate, Corning 25-035-CI (optional) 1 M HEPES, HyClone... 7.5 mL 100X glutaGRO, 7.5 mL of 7.5% Sodium Bicarbonate, 7.5 mL 1 M HEPES to 750 mL DMEM + 1 g/L glucose...Before You Start The health of the HEK293T cells is critical for optimal AAV yield. Do not overgrow your cells... layers are covered with media. 293T cells are delicate and detach very easily - media should always be...

...Complete to a 15 mL conical and an appropriate amount of 25 mM chloroquine to the conical so that the final...lentivirus can be used for a variety of downstream applications such as stable-cell line generation. Workflow...am): Harvest virus Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...10 cm tissue culture dish, Corning 430167 15 mL conical tubes, VWR 21008-216 Hydrochloric acid Sodium hydroxide... or sodium hydroxide to adjust the pH to 7.0. Typically, the solution will be basic and will need adjustment...optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI ...Start The health of the packaging cell line is critical for obtaining high viral titer. 293T cells should...