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... 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...

... 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. ...

...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 (Figure 1). 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 ...

...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 ...

...you can design a set of oligos containing your desired restriction sites and add them to your existing...overlapping oligos that once annealed can be cloned directly into the overhangs generated by restriction digest...

...Set up your water bath so that it will reach the desired temperature by the time you need it for your experiment...water bath to prevent evaporation and maintain the desired temperature. This also helps the water bath heat...

...cells Antibody Validation Using the Indirect ELISA Method Run an indirect ELISA against a purified antigen...

... likely to ligate to itself rather than to the desired insert. If you are in this situation, it is important...Additional controls are encouraged, but may only be required for troubleshooting failed ligations. The following...

...maximum possible setting on the pipette, set the desired volume of the liquid on the pipette. Depending ... you can slowly release the plunger. Remove the entire pipette from the container making sure not to touch...

...to the template DNA and the Taq polymerase, PCR requires free nucleotides [dNTPs; adenine (A), cytosine... thymine (T)] in an equal molar ratio. It also requires two unique single stranded DNA oligonucleotide...

...Last Update: February 13, 2019 Estimate of time required: ~3 h Workflow Timeline Plate set-up: 2 h qPCR...enough master mix for all samples). Each sample requires 15 μL of master mix. Pro-Tips Use a "Universal...

...recheck the pH to prevent over or undershooting the desired pH. Allow the solution to mix for 10 min and then...proceed overnight at 4 °C if needed. Transfer the entire sample to 3 x 500 mL conical bottles and centrifuge...

...plasmid DNA, the plasmid will already be in your desired bacterial strain and you will not need to obtain...

...wear glasses/goggles in addition to the BSL-1 requirements. Conclusion Although simple, following appropriate...

... in LB medium. The concentration of antibiotic required for effective over-agar selection has been empirically...

...balance and then tare it. Then add the material directly to the tube to weigh it. Make sure that your scoopula...

...linearize a plasmid to determine the size of the entire construct or excise some or all of an insert from...