Simply Cloning - Chapter 2 - PCR скачать в хорошем качестве

Simply Cloning - Chapter 2 - PCR

Simply Cloning is a video manual for making DNA constructs. Chapter 2 describes how to set up polymerase chain reaction and program a thermal cycler. Narration Script: The protocols for setting up a PCR and programming a thermal cycler are shown here and in the Protocols Poster. The Protocols Poster can be downloaded for free from the Protocols page. PCR mix: 37.5 µl ddH2O 5 µl 10X PCR buffer 4 µl dNTPs (10mM each) 1 µl Primer (forward), 10 µM 1 µl Primer (reverse), 10 µM 1 µl DNA template 0.5 µl Polymerase Using polymerase chain reaction we are going to amplify a selected region of DNA, in our case the bar gene, from our DNA template, using the pair of primers that we designed in the previous step. Let's start the PCR with programming a thermal cycler. I am going to enter a new program. The program name, as you might guess, is Bar. The reaction volume is 50 ul. PCR program: 94 oC - 2 min 94 oC - 30 sec 55 oC - 30 sec 72 oC - 1 min (or 1 min/kb of insert) Go back to 2. Repeat 29 times 72 oC - 5 min 4 oC - forever (insert 0 on most thermal cyclers) END Now let's get back to the bench and prepare a PCR mix. Here on the bench I have double distilled water, 10X polymerase buffer, dNTP mix, forward and reverse primers, DNA template, which is 50 times diluted miniprep of plasmid pFGC5941, a DNA polymerase and an empty PCR tube. Now I am going to do the pipetting: 37.5 µl of double distilled water 5 µl of 10X polymerase buffer 4 µl of dNTP mix 1 µl of forward primer 1 µl of reverse primer 1 µl of DNA template And half a microliter of polymerase Once everything is inside, I am going to mix it up by pipetting up and down. The PCR mix is ready, let's put it in the thermal cycler and run the program that we prepared today.