Dynazyme+Colony+PCR+Protocol

__**Dynazyme Colony PCR Protocol**__


 * Colony PCR Set Up ||  ||   ||   ||   ||   ||   ||   ||
 * ||  ||   || MM ||   ||   ||   ||   ||   ||
 * || 1X ||  || 11X ||   ||   |||| Conditions: ||   ||
 * Colony + water || 10 ||  || - ||   ||   |||| 95C for 3 mins ||   ||
 * Buffer 10X || 3 ||  || 33 ||   ||   |||| 94C for 30 sec || x30 times ||
 * dNTP || 0.3 ||  || 3.3 ||   ||   |||| 55C for 30 sec ||^   ||
 * For primer || 0.6 ||  || 6.6 ||   ||   |||| 72C for 1 min ||^   ||
 * rev primer || 0.6 ||  || 6.6 ||   ||   ||   ||   ||   ||
 * Dynazyme || 0.3 ||  || 3.3 ||   ||   ||   ||   ||   ||
 * water || 15.2 ||  || 167.2 ||   ||   ||   ||   ||   ||
 * || 30 ||  ||   ||   ||   ||   ||   ||   ||
 * *All quantities in μL ||  ||   ||   ||   ||   ||   ||   ||
 * For = ||  ||   ||   ||   ||   ||   ||   ||   ||
 * Rev = ||  ||   ||   ||   ||   ||   ||   ||   ||
 * For = ||  ||   ||   ||   ||   ||   ||   ||   ||
 * Rev = ||  ||   ||   ||   ||   ||   ||   ||   ||
 * Rev = ||  ||   ||   ||   ||   ||   ||   ||   ||

Note that the PCR protocol may need to be modified for very large fragments

One colony is picked with a sterile tooth pick or pipette tip and put into the PCR tube with 10uL of water. Alternatively, 1 uL of overnight culture with 14 uL of water (or just 9 uL) can be used. Heat the solution at 95 C for 5 min to break up cell walls.

This is from the excel spreadsheet attached below. The amount of primers reflect the amount for a primer concentration of 20uM. If the primer is a different concentration other than 20uM, change the volumes or dilute primer down to said concentration.

"S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Protocols\Colony PCR\Dynazyme\Colony PCR template.xls" and here "C:\kurt\storage\CIM Research Folder\DR\2012\10-26-12\colony pcr\Colony PCR template.xls"