Colony PCR

Materials:

• PCR tubes
• MQ water
• DMSO 5%
• Phusion buffer
• DNTPs
• F- and R-primer
• Phusion Polymerase
• DNA template

PCR reaction mixture:

Component	20µl	Final concentration
MQ water	up to 50µl
5x Phusion Buffer	2µl	1x
DMSO 5%	1.5µl
10mM dNTPs	1µl	200µM
primer F	1µl	0.5µM
primer R	1µl	0.5µM
template DNA	1µl	∼1ng
Phusion Polymerase 2U/µl	0.3µl	0.01U/µl

Cycling instructions:

Temperature	Time	Number of Cycles
98°C	10min	1
98°C	10sec	34
Tm-5°C	25sec
72°C	30sec/kb
72°C	10min	1
4°C	∞	1

Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/f-530s-product-information.pdf