...mammalian cells to produce antibodies Antibody Purification Purify recombinant antibodies using Protein A or ...Titering Assay for Lentivirus Quantify virus by counting antibiotic resistant colonies Generating Stable ...Video! Immunocytochemistry Use antibodies to detect antigens in cells Antibody Validation Using the Indirect...testing your virus preparations. Antibodies Protocols for common antibody applications. Intro to the Lab...lab Watch the Video! Over-Agar Antibiotic Plating Quickly add antibiotic to a pre-poured plate Watch the...Stain Determine purity and concentration of recombinant antibodies Watch the Video! Western Blot Separate ...indirect ELISA against a purified antigen to demonstrate the expected antibody dose response....

...Ampicillin resistance but is much more important for other antibiotic resistances. Plate some or all of the...containing the correct antibiotic. The resistance gene on your plasmid must match the antibiotic on the plate. .... Transformation of bacteria with plasmids is important not only for studies in bacteria but also because...both a bacterial origin of replication and an antibiotic resistance gene for use as a selectable marker.... Pro-Tips Commercial competent cells range significantly in their transformation efficiency. The lowest...amplification. Higher efficiency cells are more important if you will be transforming with very small amounts...device Reagents LB agar plate (with appropriate antibiotic) LB or SOC media Competent cells DNA you'd like...

...precipitated DNA. Supernatant contains residues, salts, and water. Pour out the supernatant in the sink. Open... other contaminants from the plasmid DNA. It is also advisable to add RNAse to the supernatant after step...Promega sell kits for isolating plasmid DNA in quantities as low as a few micrograms to as much as several...150 ng/μL to several μg/μL. The protocol below is meant to describe the general procedure for purifying ...using a kit, follow the kit's instructions. If you want to perform plasmid purification without using a ...aliquot it into several tubes/bottles. Remove the supernatant and resuspend the bacteria in buffer. Note: This...centrifugation, and remove the plasmid-containing supernatant. Add either ethanol or isopropanol to precipitate...

...concentration of the antibiotic in culture, or remove the antibiotic entirely. If the antibiotic is reduced or...different degrees of cell death upon antibiotic selection. It is important to monitor these cells regularly...dose of antibiotic, which may not be stable at 37 °C. To achieve a stable cell pool, the antibiotic selection...transfection. Some lentiviral vectors deliver mammalian antibiotic resistance (e.g., puromycin, blasticidin), which...stable cell culture after transduction. Performing antibiotic selection on transduced cells enables elimination...the transducibility of the cell line used, this antibiotic selection may be a vital step for obtaining a... Note that not all lentiviral vectors deliver antibiotic resistance. This protocol was established using...

...21bp antisense—TTTTTG 3’ Reverse oligo: 5’ AATTCAAAAA—21bp sense—CTCGAG—21bp antisense 3’ For... literature by Addgene is not meant to carry with it, and does not grant any license or rights of access...of materials by Addgene is not meant to carry with it, and does not grant any license, express or implied...protocol is provided for your convenience. See “warranty information” in appendix. Table of Contents A....Sequence of pLKO.1 TRC-Cloning Vector I.2 Recipes I.3 Warranty information Back to Top A. pLKO.1-TRC Cloning ...for cloning into pLKO.1, insert your sense and antisense sequences from step B.1 into the oligos below....below. Do not change the ends; these bases are important for cloning the oligos into the pLKO.1 TRC-cloning...

...restriction sites are present in a given sequence. You want to choose enzymes that: Do not cut within your insert...Next, we need to examine the DNA sequence that we want to amplify and design primers that will bind to ...primer design: Because we are cloning an ORF, we want to clone from the start codon (ATG) to the stop ...Product Run PCR to amplify your insert DNA. It is important to use a high fidelity taq polymerase to minimize.... The fidelity of the polymerase becomes more important the longer the expected PCR product is. You should...some DNA during the gel purification step, it is important to digest plenty of starting material. We recommend...be cut with both enzymes, and therefore it is important that the digest goes at least 4 hours and as long...

...information on nucleic acid quantification, see our protocol for DNA quantification , which can be modified...more hardy tissues such as bone, or bacteria/yeast/plant samples will require additional steps to effectively...Incubate at -20°C for 1 hour. Pro-Tips If you anticipate your RNA yield to be small, RNase-free Glycogen...minutes at 10,000 x g at 4°C and discard the supernatant. There should be a gel-like white pellet of total...minutes at 10,000 x g at 4 °C and remove the supernatant. Remove as much of the ethanol wash as possible...Air-dry the pellet for 5-10 minutes. Critical It is important to not let the pellet get too dry before resuspending...

...in the recipient plasmid. (You don't want to express the antisense version of your gene!) Are in frame ...sequence. When selecting restriction enzymes, you want to choose enzymes that: Flank your insert, but do...some DNA during the gel purification step, it is important to digest plenty of starting material. We recommend...be cut with both enzymes, and therefore it is important that the digest go at least 4 hours and as long...running a gel for purification purposes it is important to have nice crisp bands and to have space to ... your favorite gel purification method, it is important to determine the concentration of recovered DNA...DNA in a standard ligation reaction. You ideally want a recipient plasmid to insert ratio of approximately...

.... See below for options if the recombinant band makes up a significant proportion of the DNA pool. Last...Prewarm 3X LB Agar + Antibiotic plates at 37 °C. Prewarm 8X LB Agar + Antibiotic Bioassay plates. Prechill...freeze-thaw if not suspended in cryoprotectant like Glycerol or DMSO solutions. Quantify the individual Maxipreps...Amplification is usually necessary to produce sufficient quantities of library for experimental applications. Repeated...bacterial propagation (origin of replication and antibiotic selection). This recombination, at a low rate...efficiency uptake of plasmid library DNA. This quantity of cells is sufficient for libraries up to 200,000...recovery media (Lucigen, 80026-1) 8X LB Agar + Antibiotic 245 mm bioassay plates (Molecular Devices, X602...

...one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the bacteria...you will usually want to start 2 mL in a falcon tube, but for larger preps you might want to use as much...Isolating Your Plasmid DNA . Antibiotic Concentrations Commonly Used Antibiotics Recommended Concentration...times. Double check that the antibiotic in your LB media matches the antibiotic resistance on your plasmid...bacteria carrying them. The presence of an antibiotic resistance gene on a plasmids allows researchers to.... growing the bacteria in the presence of the antibiotic). Luria broth (LB) is a nutrient-rich media commonly...LB to a tube or flask and add the appropriate antibiotic to the correct concentration ( see table below...

...agarose gels, so you will want to stay in the 0.7-0.8% range if possible. You will want nice crisp bands. This...and running the gel at a lower voltage. You will want to have enough space around each band to cut without...the gel before cutting out the bands and you will want to use long-wavelength UV for as short a time as...available. Unlike the plastic tray, this will not significantly reduce the UV, but will protect the UV box from...around the band as possible. To do so, it is often important to take the excised band, lay it down on the UV...sides with the razor blade. This is especially important during the DNA purification step, as many kits...during the DNA isolation step. Finally, you will want to isolate the DNA from the gel. This is most commonly...

... to titer lentiviral preparations that confer antibiotic resistance.... to titer lentivirus preparations that confer antibiotic resistance. Determining the titer of your lentiviral...selection markers. Note this assay requires staining resistant colonies with crystal violet solution and, therefore...media as needed Days 14–18: Stain cells and count resistant colonies Equipment Class II, Type A2 Biological...lines. It is not recommended that lentiviral supernatants be subjected to multiple freeze-thaw cycles....beginning a colony formation assay, the dose of antibiotic required to kill your target cell line needs ...Treat the target cells with a range of doses of antibiotic. Determine the minimum concentration (Link opens...

...Information The final step in the construction of a recombinant plasmid is connecting the insert DNA (gene or...desired insert. If you are in this situation, it is important to treat the digested vector backbone with a phosphatase...setting up the ligation reaction itself, it is important to determine the amount of cut insert and vector...in a new window) ligation calculator to easily quantify how much vector and insert DNA to use. Combine...it will have some amount of background. What you want to see is that your vector + insert ligation has...viability of competent cells and verifies the antibiotic resistance of the plasmid Cut vector - Background...average size genes and vectors, this ratio is really meant to refer to the molarity of DNA ends available for...

...organelles, lipoproteins, and macromolecules. Importantly, iodixanol is inert and non-toxic to mammalian...protocol is composed of steps that separate out contaminants from an impure AAV preparation. The 15% iodixanol...macromolecules. The 40% and 25% steps are used to remove contaminants with lower densities, including empty capsids...iodixanol step Carefully add up to 5 mL of clarified supernatant on top of the gradient. Use 1X PBS (or formulation... to collect the solution, or you will lose a significant amount of your virus. When the first fraction.... Image adapted from Zolotukhin, S., et al. "Recombinant adeno-associated virus purification using novel...Left image adapted from Zolotukhin, S., et al. "Recombinant adeno-associated virus purification using novel...

...homology region. Hairpins in this region can significantly reduce the efficiency of two homologous ends...assembled plasmids by designing primers to split an antibiotic resistance gene to effectively create an extra...extra part, one part has half of the antibiotic gene and the adjacent part has the other half. Any colonies... should have at least the correctly assembled antibiotic gene. This trick can also enable replacement ...gel to check for size and yield. If there are significant amounts of undesired product, gel-purify DNA ... raw PCR mix can work fine in an assembly if you want to save time. Working on ice, combine segments in... product by restriction digest . Sequence the important regions of your final plasmid, particularly the...

... tub. Disinfectant may be added to prevent growth of bacteria or fungi. There are disinfectants designed...Bath 10% bleach or 70% ethanol Distilled water Disinfectant Thermometer Water bath weights and floats Reagents...allow the temperature to stabilize. Use water resistant markers, such as permanent markers, to label your...water bath, be sure to use a marker that is also resistant to chemicals such as ethanol. Writing on the tops...clean out the water bath even if you are using disinfectants as described above in step 3. You will also ...

...appropriate antibiotic) Bacterial stab Procedure Obtain an LB agar plate with appropriate antibiotic. Label...add the antibiotic resistance and your initials. Labeling within a laboratory setting is important for organization...and need to purify plasmid DNA from it, you will want to isolate an individual clonal population (single...

... save the pellet, the supernatant, or both. Conclusion Centrifuges are important tools for many different...volume as your samples. If your samples are significantly higher density than water, then you may need... The centrifuge should come up to speed with a pleasant hum. If you hear loud noises or the centrifuge...sample while the liquid portion is known as the supernatant. Depending on your experiment, you may be required...

...on ice and transfer supernatant to a sterile 500 mL bottle. Process the supernatant as follows: Filter ... Add 25 mL of PEG solution to each 100 mL of supernatant. Split into 2 x 500 mL sterile bottles as needed... at 3900 rpm for 15 min at 4 °C. Discard the supernatant and resuspend the pellets in a total of 5 mL ...now ready to purify your prep . The clarified supernatant can be kept overnight at 4 °C before proceeding...

...Background Information Bacterial glycerol stocks are important for long-term storage of plasmids. Although you...glycerol stocks of their plasmids. This way, when you want to make more plasmid DNA, the plasmid will already...be stored at 4°C for a few weeks. However, if you want to store bacteria for a longer time, you will need...completely and will improve the shelf life. It is very important that you shake the glycerol before freezing (5...