Setting up a restriction enzyme digest

Hello Everyone, I’ve created these videos primarily as instructional aides for new students, interns, and trainees in my research group. While many of the methods are generally applicable, I am of course producing these demonstrations with the materials, reagents, and equipment that are available in our lab. Also, I’ll occasionally reference the locations of items in our lab. In this video, I go over how to set up a restriction enzyme digest. Specifically, I’m setting up a PCR digest, however the procedure would be effectively the same if I was setting up a vector digest. You just want to take your DNA sample, an appropriate volume of buffer, and an appropriate amount of the enzymes. In our lab, we prefer to use NEB enzymes and accompanying buffers whenever possible, however the general skills required to set up a digest are the same regardless of what brand of reagents you use. A few notes: -- Please use this video as a guide to how to perform the mechanical manipulations for a restriction digest rather than as a protocol that provides a reliable and robust recipe that will work for all restriction digests. Evidently you will need different quantities of enzyme, buffer, and DNA in different situations. -- Generally, you want to dilute your DNA sample before putting it into a digest reaction. This is for two reasons. First, contamination with salts (such as EDTA) present in your elution can inhibit enzyme function. Second, restriction enzymes do not cut as efficiently in extremely high concentrations of DNA. As a rule of thumb, you should try to keep your DNA concentration below or close to 200 ng/uL. I did not dilute my DNA sample because during my cleanup I washed twice with PE (which contains only tris) and once with pure ethanol. From experience, I am confident that this sample is sufficiently clean. -- If I were diluting a concentrated DNA sample I would (using this particular digest as an example) add 33uL of sterile, nuclease-free water and 10uL of my DNA sample instead of just adding my DNA sample. -- While it is nice to get all of the silica out of your sample before putting it into a digest, the glass particles are inert and should not affect your reaction. -- Try to keep your enzymes on a cold block as consistently as possible. They will degrade more quickly at room temperature or if their temperature is allowed to fluctuate wildly. Removing the enzymes from the block to dispense them is fine (they won’t warm up that quickly) but you do not want to leave your enzymes on the bench the whole time you are working. -- You typically want to add less than 10% by volume of your enzymes with respect to the total reaction volume. Excess glycerol (the enzymes are stored in a 50% glycerol solution) can loosen the active sites of some restriction enzymes enough to induce star activity (non-specific cutting) or exonuclease activity. -- For guidelines on how to more precisely calculate the amount of restriction enzyme that you need, please see NEB’s “Restriction Endonucleases Technical Guide” (Google this if the following link doesn’t work) at https://www.neb.com/~/media/NebUs/Files/Brochures/RestEndo_TechGuide.pdf -- In general, we try to do a 10-fold over-digestion. This is particularly important when you’re doing vector digests, as the presence of even a tiny quantity of undigested DNA will result in a lot of background colonies when you transform your ligation mixture. -- Make sure to mix your digest thoroughly. If the enzymes (which are stored in 50% glycerol, which is heavier than water) are allowed to sit at the bottom of the digest, your digest will likely not get complete digestion. -- I let my PCR digests go for about 3 hours at 37 degrees C (for reactions where the enzymes exhibit optimal activity at 37 degrees C). About me: I am currently (at the time of uploading this video) a doctoral student in the Chemical and Biomolecular Engineering department at The Ohio State University. My advisor is Dr. David Wood, and our research group specializes in protein engineering and downstream processing for the biotechnology industry. I’m also very new at producing instructional videos so I apologize in advance if these are not of professional quality. For instance, in my first few videos I did not know how to get rid of the timestamp (which is wrong), however I posted them anyways. Please keep comments civil and apolitical. You are of course welcome to ask questions, however it is unlikely that I will answer them if you are not a member of my research group. Thanks for watching, Steven