Team:Groningen/protocols/PCR

PCR

Materials:

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

PCR reaction mixture:

Component	50 µl	Final concentration
MQ water	up to 50 µl
5x Phusion Buffer	10 µl	1x
DMSO 5%	1.5 µl
10 mM dNTPs	1µl	200 µM
primer F	2.5 µl	0.5 µM
primer R	2.5 µl	0.5 µM
template DNA	1 µl	∼1 ng
Phusion Polymerase 2 U/µl	0.3 µl	0.01 U/µl

Cycling instructions:

Temperature	Time	Number of Cycles
98°C	2 min	1
98°C	10 sec	34
Tm-5°C	25 sec
72°C	30 sec/kb
72°C	10 min	1
4°C	∞	1

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