...double digest (digesting with two enzymes at the same time), you will need to determine the best buffer that...and the amount of DNA in the reaction, incubation time can range from 45 mins to overnight. For diagnostic... of methylation sensitive restriction sites . Sometimes enzymes cut sequences which are similar, but not...amount of Master Mix to each tube. This will save you time and ensure consistency across the reactions....

...the same time as the virus-mediated transduction. For some cell lines, transduction is optimized if the ...cannot be used for later experiments. Workflow Timeline Day 0: Seed and transduce cells Day 2: Replace...that case, polybrene should only be added at the time of viral transduction, and not during the cell seeding...

...column gradient for AAV purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration... a T70i rotor at 10 °C. Pro-Tip If you need more time, you can alternatively centrifuge for 2 h at 200,000...Repeat for each QuickSeal tube. (Optional) For first time users, it is a good idea to assay each fraction ...sample and make sure to pipet back and forth a few times to mix the iodixanol that has settled at the bottom...

...through the use of a selection curve to determine optimal antibiotic concentration. Last Update: Oct. 27,...30 minutes with the lid on to give the antibiotic time to more fully absorb. During the incubation, transform...Shown below are the results from an experiment optimizing the concentration of carbenicillin, plated over-agar...

...liter. Place a thermometer in the water bath. Oftentimes, the water bath will have a spot to secure the...that it will reach the desired temperature by the time you need it for your experiment. Water baths tend... the water bath, place the lid back on. Note the time that you’ve put your materials in the water bath...

...protective equipment (PPE) and wear it the whole time you are working in the lab. Do not eat, drink, chew...supervisor what is available and the appropriate contact times for each agent. Make sure you have enough space ...laboratory. Don’t mouth pipette! There may be some times when you may be working with a protocol that requires...

...magnifies the image four times the actual size, the 10x objective magnifies it 10 times, and so on. The ocular...focus, use the stage arm to move the stage (and ultimately your slide) until your field of view is centered...

...approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration... use long-wavelength UV and expose for as little time as possible to minimize damage to the DNA. Note:...the gel at a lower voltage for a longer period of time; b) using a wider/thinner gel comb; or c) loading...

... of Solution II and invert the tube carefully 5 times to mix the contents. The contents will become clear...Solution III to each tube. Mix by inverting several times. A white precipitate will be formed which contains... 0.1 mM EDTA). Pro-Tip DNA resuspension can take time, it is a good idea to let it sit for several hours...

...feet. Pro-Tip If they are able, sometimes scientists work for a long time while standing. Wear comfortable...

...it through a flame, just be sure to allow enough time for the loop to cool before touching it to the bacteria...often true for large unstable plasmids, which sometimes recombine at 37 °C. Be sure to check this before...

...will want to use long-wavelength UV for as short a time as possible to get the bands cut out. Once you have...the gel at a lower voltage for a longer period of time; b) using a wider gel comb; or c) loading less DNA...

...weigh out 100 g three times, placing the sucrose in your container after each time. Place the weighing ...

...Independent Cloning (LIC) obviates the need for the time-consuming ligation step of traditional cloning methods...sterile water (instead of TE buffer) to ensure optimal salt concentrations in subsequent reactions. Step...

...place, and the control panel where you can set the time and speed needed for your experiment. Procedure ...Adjust the centrifuge settings according to speed and time (and temperature, if applicable) listed in your ...

... institution’s biosafety regulations. Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells ...with multiple integration events leading to underestimation of the true titer. Calculate the transduction...

...can work fine in an assembly if you want to save time. Working on ice, combine segments in the Gibson ...rate when assembling more than five fragments at a time. Resources and References New England Biolabs (NEB...

... making a copy of a piece of DNA and at the same time adding restriction sites to the ends of that piece...both function in the same buffer, as this will save time later. In our example, we will use EcoRI and NotI...

... the absorbance of a liquid you can accurately estimate the concentration of a substance in that liquid...

...on a nucleotide when adding a nucleotide one at a time. The main property of primers is that they must ...