...Calculate the average absorbance of the duplicate samples on the plate. Subtract the average absorbance ...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...intended to support reproducibility in science. General Guidelines Acrylamide percentage varies in SDS-...depending on the size of your cell pellet but will generally be between 250–1000 µL . The ideal lysis buffer...TBST at RT on a shaking platform. Dilute the horseradish peroxidase-conjugated secondary antibody to the...

...L” shape, and then used like a cell spreader. Several other devices may be used for this purpose, provided...your petri plate. Incubate the plate at room temperature for at least 30 minutes with the lid on to give.../mL Carbenicillin plated over-agar Plate shows several individual colonies and effective selection. 150.../mL Carbenicillin plated over-agar Plate shows several individual colonies with smaller size than the ...plate). Please note we have found that there is generally a broad range of antibiotic concentrations that...

... to room temperature. Now screw on the top of the bottle and store the LB at room temperature. When ready...bacteria since they will grow more slowly at lower temperatures. After incubation, check for growth, which is...plasmids should only need to be grown for 12-16 hr on average. Certain features of a plasmid may render it low...agar plate before growing in liquid culture. More aeration may help to increase the density of the culture...

...reactions involve DNA fragments that have been generated by restriction enzyme digestion . Most restriction...depicts the ligation of two sticky ends that were generated by EcoRI digestion: Usually, scientists select...for details on optimization. Incubate at room temperature for 2hr, or at 16°C overnight (following the ...using "high concentration" ligase, 5min at room temperature is enough. For trickier ligations (such as ligation...cloning. While 3:1 will get you in the ballpark for average size genes and vectors, this ratio is really meant...

...iodixanol step has 1M NaCl to destabilize ionic interactions between macromolecules. The 40% and 25% steps...prep with full (genome containing) particles. On average, iodixanol-purified AAV preparations contain ~20%...methods improves infectious titer and yield." Gene therapy 6.6 (1999): 973-985 (Link opens in a new window... Rist Wolfgang, and Lamla Thorsten. Human Gene Therapy Methods. August 2015, 26(4): 147-157. doi:10.1089...

...tubes 96-well optical plate Pipette tips General Considerations Always run standards and samples in duplicate... master mix which contains a high-quality DNA polymerase and a blend of dTTP/dUTP to minimize carryover...Prepare a plasmid stock of 2 x 10 9 molecules/μL to generate a standard curve: One option is to use plasmid... be seen. The presence of a second peak at a temperature of ~70–75 °C usually indicates the presence of...

...change in a plasmid (such as point mutations). Generate DNA segments by PCR. Run PCR product on an agarose...anneal to each other. Phusion High-Fidelity DNA Polymerase - incorporates nucleotides to “fill in” the gaps...PCR products, one method is to “stitch” together several oligos. This technique is especially useful for...2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods , 6(5), 343–345...

...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...platform. Permeabilize cells for 10 min at room temperature ( RT ) on a rocking platform in 500 µL permeabilization... optimal antibody concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL of the diluted... optimal antibody concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL fluorescently-labeled...

...Use this protocol to generate lentivirus...downstream applications such as stable-cell line generation. Workflow Timeline Day 0: Seed 293T packaging...batch of 1 mg/mL PEI and for each cell line. Considerations Before You Start The health of the packaging...The viral supernatant can be stored at 4 °C for several hours but should be aliquoted and snap frozen in...

...annealed can be cloned directly into the overhangs generated by restriction digest of existing sites in the... additional bases to complement the overhangs generated when digesting the vector with EcoRI and SalI ...bench top) allowing for slow cooling to room temperature (~45 minutes). Method #2 Place mixed oligos in...

...downstream applications. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell...2.5x10 6 TU/mL For a more accurate titer, take the average of multiple dilutions. Sample Data Figure 1: 293T...

...a blunt end ligation. Use T4 DNA Polymerase or Klenow DNA Polymerase I for 3′ overhang removal and 5′ ...gently by pipetting. Incubate tube at appropriate temperature (usually 37 °C) for 1 hour. Always follow the...

... chemical or electrical based transformation, generating what are commonly referred to as 'competent cells... from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator...This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded in ...

...using ImageJ software and a standard curve is generated. The ratio of the antibody protein content to ...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...relative intensity of the protein bands to the overall lane. Import the gel image into ImageJ. Select ...

...Digest of Plasmid DNA Background Information During several different stages of molecular cloning, it is important...ratio of nucleic acid to protein (260/280) is generally used as an indicator of the purity of DNA samples...

... appropriate temperature. Growth conditions, including copy number and growth temperature, can be found...

... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell...,000 TU/mL For a more accurate titer, take the average of multiple dilutions. Sample Data Figure 1: A549...

...look at the side of an objective, you will see several pieces of information, but the most important (...your observations. Some microscopes have digital cameras attached to them that allow you to capture images...

...gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified... gel, make 10% more volume than needed because several microliters can be lost in pipetting. For example...

... gel, make 10% more volume than needed because several microliters can be lost in pipetting. For example...