"
Team:Wisconsin-Madison/notebookprotocols
From 2011.igem.org
| Line 212: | Line 212: | ||
<strong><font size="3"> | <strong><font size="3"> | ||
| - | + | PCR Amplification | |
</font></strong><p> | </font></strong><p> | ||
| + | If you want to obtain a gene from genomic DNA, you will use PCR Amplification on the Thermal Cycler with primers that flank the gene of interest. See Making Primers section to see how to make primers. | ||
| + | One of the first things when you receive the primers is to do a gradient around the calculated Tm (Tm+3 is using Phusion enzyme). This is to find the optimal Tm temperature for high amplification of the gene. For example, if the calculated Tm is 55C, then the gradient range will be from 49 to 59 in increments of 1 or 3 degrees depending on how exact you want it. (I did 3 degree increments and chose the one that had the brightest band) | ||
Revision as of 15:59, 8 August 2011
Notebook >> Overview, Members, Advisers, Sponsors
|
PCR Amplification
If you want to obtain a gene from genomic DNA, you will use PCR Amplification on the Thermal Cycler with primers that flank the gene of interest. See Making Primers section to see how to make primers. One of the first things when you receive the primers is to do a gradient around the calculated Tm (Tm+3 is using Phusion enzyme). This is to find the optimal Tm temperature for high amplification of the gene. For example, if the calculated Tm is 55C, then the gradient range will be from 49 to 59 in increments of 1 or 3 degrees depending on how exact you want it. (I did 3 degree increments and chose the one that had the brightest band) |
