"
Team:Wisconsin-Madison/notebookprotocols
From 2011.igem.org
| Line 216: | Line 216: | ||
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. | 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) | 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) | ||
| + | <p><br> | ||
| + | |||
| + | <strong><font size="3"> | ||
| + | PCR Amplification Protocol | ||
| + | </font></strong><p> | ||
| + | Below is a standard protocol for amplifying a gene from chromosomal or plasmid DNA. The reagents used can change depending on the GC content of the template DNA (DNA that will be amplified). For example, if GC content exceeds (60 to 70%) then DMSO is added and 5X Buffer GC is used. If its below this range, then use 5X Buffer HF without DMSO. | ||
| + | <p><br> | ||
Revision as of 16:00, 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)
Below is a standard protocol for amplifying a gene from chromosomal or plasmid DNA. The reagents used can change depending on the GC content of the template DNA (DNA that will be amplified). For example, if GC content exceeds (60 to 70%) then DMSO is added and 5X Buffer GC is used. If its below this range, then use 5X Buffer HF without DMSO.
|
