Setting up multiple PCR reactions from a mastermix скачать в хорошем качестве

Setting up multiple PCR reactions from a mastermix

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. Note that I'm setting up Q5 PCR reactions. The recipes for a PCR using Q5 DNA Polymerase can be found on the NEB web page. In this video, I go over how to set up multiple PCR reactions from a mastermix. This is a useful time-saving habit to develop if you are setting up multiple reactions of any type that are mostly similar. For example, in this video I am setting up 6 Q5 PCR reactions where everything is the same except for the reverse primers and the template. I prepare 6.2uL reactions worth of mastermix to account for pipetting losses; this is a good habit to get into so you don’t run out of material for your last tube. When setting up a PCR, you can prepare a variety of different “mastermixes” depending on what you’re trying to do. You might prepare a mastermix where everything is the same except for the template, like you would do for a colony PCR screen. Alternately, you might prepare a mastermix where both primers are different, but the template is the same (like if you were trying to clone a gene into several different plasmids). You might also prepare a mastermix that contains no primers or template. You might do this if you were preparing several PCR reactions of the same type (e.g. Q5 PCR) that did not use the same template or primers Of course, I might conceivably be doing a series of restriction enzyme digests with NdeI and HindIII-HF, like one would do for digest checks on new clones. When I do a single digest check, I typically (depending on the concentration of the plasmid) mix 5uL of plasmid DNA, 12.6uL water, 2uL of CutSmart, and 0.2uL of each enzyme (for a 20uL total reaction volume). If I needed to do 20 digest check with the same enzymes, I would mix 252uL (=20*12.6uL) of water, 40uL (=20*2uL) of CutSmart, and 4uL (=20*0.2) of each enzyme in a single tube. I would then aliquot 15uL of this digest mastermix out into 20 different tubes and then add 5uL of plasmid sample to each tube. 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