Dynazyme Colony PCR Protocol

2015-01-13

azim58 - 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














































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"