Dynazyme Colony PCR Protocol

2015-01-13

azim58 - Dynazyme Colony PCR Protocol

Colony PCR Set Up

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

*All quantities in μL

For =

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"

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