Team:Cambridge/Protocols/PCRcolony

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Revision as of 10:44, 18 July 2012

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Colony PCR:

Used to amplify DNA directly from cell culture, the reaction is the same as for normal PCR but with modified reaction composition and cycle settings, here shown for a 50 µl reaction using Taq polymerase.

Reagent	Volume (µl)	Final Concentration
Water	35.7
10 mM dNTPs	1	200 µM
10 x NH₄ buffer	5	1x
Forward Primer	2.5	0.5 µM
Reverse Primer	2.5	0.5 µM
Template Cells	1.3 (from liquid culture or picked colony
Taq polymerase 5u/µl	1	0.1 u/ µl

PCR machine settings:

		Temperature (oc)	Time (s)
Step 1 (cell breakage)		95	360
Step 2 (Cycle 30x)	Denaturing	98	10
	Annealing	60	30
	Elongation	72	120
Step 3 (final extension)		72	300

Safety considerations: It is important to observe correct laboratory procedure and wear appropriate clothing and gloves. PCR occurs at high temperature and this may present a risk, depending on the PCR machine employed. For handling the cell culture, appropriate measures should be in place to deal with biohazardous waste.

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@@ Line 24: / Line 24: @@
 ||10 mM dNTPs||1||200 µM
 |-
-||10 x NH4 buffer||5||1x
+||10 x NH<sub>4</sub> buffer||5||1x
 |-
 ||Forward Primer||2.5||0.5 µM