Revision as of 12:08, 29 July 2014

PCR for bacterial culture

1. In a cold PCR tube (Eppendorf), pipet in the presented order the solutions listed in Table 1.


Component	Initial concentration	Final concentration	Example for 50μl
H₂O	-	Add to final volume	27.75μl (50μl final)
Tp Green 5X	5X	1X	10μl
dNTPs	10mM	200μM	1μl
MgCl₂	-	-	4μl
Primer A	1mM	10μM	2μl
Primer B	1mM	10μM	2μl
Culture	-	-	2μl
GoTaq DNA polymerase	-	-	0.25μl
DMSO	-	-	1.5μl

Note: enzyme is added last, and dNTP second from last. 48μl of mixture is inserted in a PCR tube before adding 2μl of culture.

2. Program the PCR machine according to the Table 2.


Cycle step	Temperature	Time	Cycle
Initial denaturation	95 - 98°C	30 s - 3 min	1
Denaturation	95 - 98°C	10 - 30 s	25 - 30
Annealing	variable	30 s	25 - 30
Extension	72°C	30 s - 2 min	25-30
Final extension	72°C	10 min	1
Final extension	4 - 8°C	hold	1

3. Verify correct amplification by agarose gel electrophoresis.

@@ Line 60: / Line 60: @@
 | 1.5μl
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+<span style="color:red">Note: enzyme is added last, and dNTP second from last. 48μl of mixture is inserted in a PCR tube before adding 2μl of culture.</span>
 :2. Program the PCR machine according to the Table 2.