...Validation Using the Indirect ELISA Method Antibody Validation Using the Indirect ELISA Method You may...This protocol describes how to run an indirect ELISA against a purified antigen to demonstrate the expected...Introduction This protocol describes how to run an indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) against...antibody and an isotype control antibody to the desired concentrations in the antibody dilution buffer....HRP-conjugated isotype-specific secondary antibody to the desired concentrations in the antibody dilution buffer....Check the plate periodically to determine when the desired color change has occurred. The color will change... to optimize experimental conditions. When the desired color change has occurred, gently remove the plate...

...Sharps container Biohazard waste container Fire blanket Fire extinguisher Guidelines BSL-1 Guidelines Before...eyewash station, safety shower, fire blanket, and extinguisher are also required to be present in the room....each level, as each level has different safety requirements. BSL-1 is designated for those working with ...times when you may be working with a protocol that requires shaking or mixing, which may produce aerosols ... PPE protocols . Working in a BSL-2 laboratory requires laboratory glasses in addition to coat and gloves...room and on the door. BSL-2 centrifugation steps require an aerosol-tight lid and rotors should be loaded...in a biosafety cabinet. BSL-2 safety protocols require bloodborne pathogens training. It is strongly recommended...

...stringent buffers and a sonication step may be required for hard to extract proteins such as those in ...standard curve. Determine the volume of sample required to load an equivalent amount of total protein ...continue running the gel until you have obtained the desired separation. Gently open the gel with the spatula...Device. Close the lid of the device. Select the desired method and make sure the parameters are correct... incubation Dilute the primary antibody to the desired concentration in blocking buffer. Pro-Tip The optimal...peroxidase-conjugated secondary antibody to the desired concentration in blocking buffer. Pro-Tip The optimal...

... as bone, or bacteria/yeast/plant samples will require additional steps to effectively lyse open the cells...wash and add RNase-free water or TE as soon as the entire tube is dried but while the white pellet is still...Pro-Tip To avoid multiple freeze-thaw cycles of your entire RNA sample, consider making smaller aliquots of...

...possible but note that individual libraries may require modifications dictated by the originating laboratory... can be scaled according to the actual number. Required Quality Control (QC) Pooled libraries can be challenging...a smaller plasmid containing only the elements required for bacterial propagation (origin of replication...100,000 plasmids. Colonies may appear small and require extra incubation time in order to be enumerated...colonies but as long as one can ensure more than the required colonies are present. If colonies are not present...can dramatically plateau and sometimes fall off entirely if the column or reagents are significantly overloaded...

... RNAi Consortium has built a library of shRNAs directed against 15,000 human and 15,000 mouse genes. Addgene...shRNAs. pLKO.1 can be introduced into cells via direct transfection, or can be converted into lentiviral...protein expression, you may transfect plasmid DNA directly into the target cells. The shRNA will be expressed...OPTI-MEM necessary given that each reaction will require 6 μL FuGENE® + 74 μL OPTI-MEM. For example: 1x ...polypropylene tube, add OPTI-MEM first. Pipette FuGENE® directly into the OPTI-MEM – do not allow FuGENE® to come...for a total volume of 100 μL. Pipette master mix directly into the liquid and not onto the walls of the ...

...verify the ends of the insert. Longer genes may require internal primers that anneal to a specific region...portion of the insert, but we do not sequence the entire plasmid. Addgene strongly recommends that you...Make sure the primer only anneals once within the entire construct. Is the primer an appropriate distance... within the Addgene sequencing results? If you require additional sequencing and need to design a custom...

...bottle may explode!) and then loosely cover the entire top of the bottle with aluminum foil. Autoclave...shaking incubator. Note: Some plasmids or strains require growth at 30°C. If so, you will likely need to ...in the media (see right). Notes: Some protocols require bacteria to be in the log phase of growth. Check...bacteria incubated at 30°C rather than 37°C often require longer incubation times. Double check that the ...

... in 1.5 mL microfuge tubes at 10,000 rpm would require a tabletop microcentrifuge like the one in the ... you are working. Even if your samples may not require specific PPE, you never know what else has been...RPM), these are not the same units and are not directly interchangeable. If you need to convert between...supernatant. Depending on your experiment, you may be required to save the pellet, the supernatant, or both. ...

...cell lines and selection markers. Note this assay requires staining resistant colonies with crystal violet...colony formation assay, the dose of antibiotic required to kill your target cell line needs to be empirically...minimum concentration (Link opens in a new window) required to kill all of the cells. Use this dose for the...cell suspension into each well of the 6-well dish directly on top of the virus. Swirl gently to mix and place...

..., but do not cut within your insert Are in the desired location in your recipient plasmid (usually in ...possible to use a single enzyme, but this will require phosphatase treatment of your recipient plasmid... fear. You have other options, such as: Adding desired restriction sites to flank your insert : You can...restriction enzymes that cut within your insert. Adding desired restriction sites to your recipient plasmid : You...

... per well. Allow the HeLa cells to grow to the desired density before labeling. Section 2: Fixing and ... container. Dilute the primary antibody to the desired concentration in antibody dilution buffer. Pro-...fluorescently-labeled secondary antibody to the desired concentration in antibody dilution buffer. Pro-...

... science-fiction levels of detail. You can take entire courses on microscopy and still have more to learn...nosepiece so that the lowest power objective is directly over the stage. It is best practice to start with...attached to them that allow you to capture images directly to a computer, but many microscopes do not and...

...Introduction Many molecular biology techniques require highly purified and concentrated plasmid DNA. This... volume of bacteria to grow (low copy plasmids require larger cultures). Centrifuge the culture to pellet...proceeding with DNA preparation. Pro-Tip If your entire overnight culture cannot fit into a single centrifuge...

...-agar 1 L Sterile H 2 O Sterile plates of your desired size - we usually use 60 mm x 15 mm plates which...next to the flame and begin pouring. Measure your desired amount of agar with a pipete for the first plate...particular plate. For the remainder of the plates, pour directly from the bottle. Pro-Tips Be sure to swirl your...

...that: Do not cut within your insert. Are in the desired location in your recipient plasmid (usually in ...amplified (the ORF in this case), not the Tm of the entire primer. If you are amplifying from a plasmid or...plenty of starting material. We recommend using your entire PCR reaction and 1μg of recipient plasmid. It is...

...Spectrophotometer to see if the sample has reached the desired concentration. If the sample concentration is still...volume of the sample is reduced enough to reach the desired concentration. Pro-Tip Periodically check the concentration...Spectrophotometer to see if the sample has reached the desired concentration. Gently transfer the sample from ...

...generally considered biosafety level 1 but may require BSL-2 handling depending on the insert. Please ...liquid up and down 10–20 times. Gently cover the entire dilution plate with Microseal adhesive seal - do...droplets are not disrupted, insert the pipette tips directly in the center of the well and tilt to a 45° angle...

...bacterial colonies. Equipment Sterile toothpicks or wire loop Bunsen burner (or other small flame source)... top of the glycerol stock. Pro-Tip If you use a wire loop you can sterilize it by passing it through ...

...and with a clean, sterile razor blade, slice the desired DNA fragment from the gel. Notes: To protect the...with the gel fragment. The weight of the gel is directly proportional to its liquid volume and this is ...