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...tip is changed between samples. Tips can come loose in a bag, or can come preloaded into tip boxes. In either...and any changes to the amount you are dispensing can negatively impact your experimental results. This...come in often indicate a volume range that the tip can hold. This should give you an idea of what pipette...purpose of the pipette tip is so that the same pipette can be used for measuring different samples without cross...case, once assembled into their boxes, pipette tips can be sterilized in an autoclave as needed to prevent...the liquid. Once you are clear of the liquid you can slowly release the plunger. Remove the entire pipette...

...storage of the bacteria, you can proceed with creating a glycerol stock . You can now isolate your plasmid... Inoculating a Liquid Bacterial Culture Plasmids can carry one or more antibiotic resistance genes, which... the liquid LB + antibiotic and swirl. Note: You can also use a long toothpick to select the colony, swirl...Unless otherwise indicated, the antibiotic powder can be dissolved in dH 2 0. Addgene recommends making...approximately 18–30 h). On the other hand, smaller plasmids can be present in large numbers, 50 or more per cell,...

...Solid BSL-2 waste can be collected in designated biohazardous waste containers that can be autoclaved. Conclusion...designated sharps container. Sharps include anything that can pierce your skin, including, but not limited to broken... be used for proper disposal. Liquid BSL-2 waste can be decontaminated in a final concentration of 10%...administrators, or anyone else associated with Addgene, can be held responsible for your use of the information...decontaminate as necessary. For large spills that cannot be easily cleaned, evacuate and close off the area...

... single copy of DNA (the template), a researcher can create thousands of identical copies using a simple...bacterium, Thermus aquaticus (Taq) . Taq polymerase can withstand many heating and cooling cycles, which ...synthesis. After the PCR is complete, the product can be verified based on size by gel electrophoresis ...template DNA for all your reactions, the Template DNA can be added to the master mix. Forward and Reverse Primers...DNA synthesis is ~1-2 kb/min. The extension time can be adjusted according to the length of the target... 2+ stabilize the buffer solution. These cations can also be used for PCR-mediated DNA mutagenesis. A ... Taq DNA polymerase: Special DNA polymerase that can withstand radical temperature changes during a typical...

...’t have access to a refrigerated centrifuge, you can carefully bring a centrifuge into a cold room for...tip in the tube. Precipitate your sample(s). You can use either Isopropanol or Lithium Chloride for this..., instead of incubating at -20°C for 1 hour, you can try incubating at -80°C overnight. Centrifuge for... see our protocol for DNA quantification , which can be modified for RNA. Store your RNA sample(s) at ...tip in the tube. Precipitate your sample(s). You can use either Isopropanol or lithium Chloride for this...

... 50 µL reaction in 1 hour. Using this ratio, you can calculate the minimal amount of enzyme for your reaction...the amount of DNA in the reaction, incubation time can range from 45 mins to overnight. For diagnostic digests...after removal from the -20 °C freezer because heat can cause the enzymes to denature and lose their function...recognition sites. This is due to "Star Activity" and can happen for a variety of reasons, including high glycerol...enzyme(s) (for instance, in a diagnostic digest), you can create a "Master Mix" consisting of all of the reaction...) NEB's compatible cohesive ends chart . If you cannot find compatible sticky ends, you will need to fill...

..., you can weigh out the reagents in batches. For example, if you need 300 g of sucrose, you can weigh ... laboratory technique, as imprecise measurements can affect the outcome of your experiment. In this protocol...Now that you have your reagents weighed out, you can begin making your solutions!...

...transform bacteria so that you can grow up more of the plasmid) you can save a lot of time by shortening...genetic modifications to create bacterial strains that can be more easily transformed and that will help to ...Incubate plates at 37°C overnight. Tips and FAQ How can I save time when carrying out transformations? If...transforming large plasmids (>10 kb) or BACs, what can I do? Chemically competent cells are fast and easy.... Pro-Tips Commercial competent cells range significantly in their transformation efficiency. The lowest...

...accurate reading of DNA concentration and purity. This can be done with a spectrophotometer. A spectrophotometer...absorb. By measuring the absorbance of a liquid you can accurately estimate the concentration of a substance...NanoDrop, which is a very small spectrophotometer that can accurately read DNA concentration and purity in as...the NanoDrop, if two consecutive samples have significantly different concentrations, it is possible that...between samples if they are expected to vary significantly in concentration. DNA dissolved in water is ...

...the pellet which can cause problems with some common reactions. Air dry the pellet (can be done by inverting...perform plasmid purification without using a kit, you can find a protocol for kit-free plasmid mini-prep at...sample. Pro-Tip Water-saturated phenol-chloroform can be used if TE-saturated is not available. Vortex ...attached to the tube after the 70% ethanol wash. You can also pipet the supernatant out of the tube if you...Tris-HCl pH 8, 0.1 mM EDTA). Pro-Tip DNA resuspension can take time, it is a good idea to let it sit for several...preparation. Pro-Tip If your entire overnight culture cannot fit into a single centrifuge tube, aliquot it into...

...you can easily scan the plate for individual cells, which should be on the same focal plane. Scan the ...the microscope later when scanning the plate for individual cells, which can be difficult to find. Leave...calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) Trypsin ...this protocol. Conditioned medium (details below) can be used to enhance the growth of cells in individual...protocol has been optimized for adherent cells but can be modified for suspension cells. The number of monoclonal... that the cells have detached from the plate and can be isolated. When trypsinized, detached cells will...cells for 13 days. Note that growth rate differences can be inferred by the differences in colony size. Figure...

...primer, one can include more than one nucleotide at that position termed a mixed site. One can also use ...completely to the template DNA strand so elongation can proceed. Usually a guanine or cytosine is used at...

...wearing open-toed shoes, cloth coverings or “booties” can be used to protect your feet. Pro-Tip If they are...Eye protection such as goggles and/or face shields can be used as needed. For BSL-2 work always wear glasses...administrators, or anyone else associated with Addgene, can be held responsible for your use of the information...be snug to your hand, but not so tight that you cannot perform your duties. Be sure to try out different...

...attached to the backbone and the complete plasmid can be transformed into bacterial cells for propagation...Whenever you need to set up ligations in the future you can thaw a new tube that you know has only been thawed...ligation of annealed oligos) the efficiency of ligation can be improved by incubation at 37°C. Proceed with bacterial...insert to vector ratio is usually sufficient, you can optimize the amount of insert and vector to improve...If the DNA concentrations are low such that you cannot get all 100ng of DNA, buffer and ligase into a ...

...reproducibility in science. General Considerations The ELISA can be completed in a single day or broken up over several...best concentration for your assay. Washing steps can be done by hand but if available, use an automated... to add 200 µL wash buffer to each well. Washing can be done by hand but if available, use an automated...

...glycerol stock. Pro-Tip If you use a wire loop you can sterilize it by passing it through a flame, just ...angle, the way you would hold a pencil, so that you can make a broad stroke. Only touch the surface of the...single colonies. Once you have single colonies, you can proceed to Recovering Plasmid DNA or use the individual...

...Introduction This protocol can be used to purify AAV of any serotype. In medicine, iodixanol can be used as an intravenous...rotor at 10 °C. Pro-Tip If you need more time, you can alternatively centrifuge for 2 h at 200,000 x g at...used to collect fractions from the gradient. You can collect fewer fractions once you have a good idea... 3 mL collected corresponds to the 60% phase and can be discarded. The fractions obtained from the 40%... to collect the solution, or you will lose a significant amount of your virus. When the first fraction...

...A wide variety of protocols from Addgene that can be used for basic molecular biology, plasmid cloning...page you'll find practical lab protocols that you can use for a wide range of applications, with videos...for select protocols in the right-hand column. You can find even more video content on the Addgene Video...

... the recombinant antibody can be used as-is or the recombinant antibody can be purified from the supernatant...transfection and expression, the recombinant antibody can be purified for use in a variety of applications....supplement the flask with 4% BCD Feed. Pro-Tip The feed can be repeated up to 4 times for a total of 16% of the...Pro-Tip The optimal ratio of DNA:PEI may vary significantly and should be empirically determined for your...

...expression of shRNAs. pLKO.1 can be introduced into cells via direct transfection, or can be converted into lentiviral...plasmid for producing viral particles. Note: pLKO.1 can also be used with packaging plasmid pCMV-dR8.2 dvpr...Biomedical Research hosts an siRNA Selection Program that can be accessed after a free registration. Guidelines...pLKO.1 vector was created. Additional design rules can be found at the Broad Institute's TRC shRNA Design...that two different shRNAs that target the same gene can produce the same phenotype will alleviate concerns... on 0.8% low melting point agarose gel until you can distinctly see 2 bands, one 7kb and one 1.9kb. Cut...is for transfection in a 6 cm plate. The protocol can be scaled to produce different amounts of virus as...