Team:Cambridge/Protocols/Colony PCR

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==Colony PCR==
==Colony PCR==
-
protocol description
+
PCR can be used to amplify DNA directly from cell culture. We used this as a diagnostic tool to check that our constructs were successful. We used the standard PartsRegistry sequencing primers, [http://partsregistry.org/wiki/index.php?title=Part:BBa_G00100 VF2] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_G00101 VR], and ran the PCR product on a gel to check the length.
===Theory===
===Theory===
-
How it works
+
Exactly the same as normal PCR except that the template consists of whole cells -- a colony picked from a plate or a small volume of liquid culture might be used -- and there is an initial heating step to lyse the cells.
 +
 
===Practice===
===Practice===
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Composition of 50 μl reaction:
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Proceed as in normal PCR except with this modified reaction composition (for a 50μl reaction) and cycle settings:
-
{|
+
 
-
|water
+
{|border="1px" align="center" style="text-align:center;"
-
|35.7 μl
+
!Name
 +
!50 μl reaction
 +
!Final concentration
|-
|-
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|10 mM dNTPs
+
|Water (reverse osmosis) || 35.7 μl ||
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|1 μl
+
|-
|-
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|10× NH4 Buffer
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|10mM dNTPs || 1 μl || 200 μM each
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|5 μl
+
|-
|-
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|50mM MgCl2
+
|10× NH4 buffer || 5 μl || 1×
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|1 μl
+
|-
|-
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|forward primer
+
|Forward Primer 10 μM|| 2.5 μl || 0.5 μM
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|2.5 μl
+
|-
|-
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|reverse primer
+
|Reverse Primer 10 μM || 2.5 μl || 0.5 μM
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|2.5 μl
+
|-
|-
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|liquid culture
+
|Template cells || 1.3 μl liquid culture or a picked colony ||
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|1.3 μl
+
|-
|-
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|Taq polymerase
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|Taq 5u/μl || 1 μl || 0.1 u/μl
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|1 μl
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|}
|}
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Settings of PCR machine
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Settings of PCR machine:
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{|
+
{|border="1px" align="center"
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|Step 1
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|'''Step 1''' (cell breakage)
|95°C
|95°C
|6 min
|6 min
|-
|-
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|Step 2
+
|'''Step 2''' (cycle)
|98°C
|98°C
 +
55°C
 +
 +
72°C
|10 s
|10 s
 +
30 s
 +
 +
180 s
|-
|-
-
|
+
|'''Step 3''' (final extension)
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|55°C
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-
|30 s
+
-
|-
+
-
|
+
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|72°C
+
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|3 min
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-
|-
+
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|Step 3
+
|72°C
|72°C
|5 min
|5 min
|}
|}
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How to do it in the lab
 
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{Standard layout for procedures is to use: 
 
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*'''''<Procedure title - aka what you are doing>'''''
 
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:# <step 1>
 
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:# <step 2>
 
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:#* '''<additional notes/important information regarding the previous step>'''
 
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the text within the < > is what should be written, don't include < > in actual writeup :P
 
-
 
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if in doubt see the gel electrophoresis protocol
 
-
 
-
}
 
===Safety===
===Safety===
-
The safety implication of the procedure.
+
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.
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}

Revision as of 10:06, 14 September 2011

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Contents

Colony PCR

PCR can be used to amplify DNA directly from cell culture. We used this as a diagnostic tool to check that our constructs were successful. We used the standard PartsRegistry sequencing primers, VF2 and VR, and ran the PCR product on a gel to check the length.

Theory

Exactly the same as normal PCR except that the template consists of whole cells -- a colony picked from a plate or a small volume of liquid culture might be used -- and there is an initial heating step to lyse the cells.

Practice

Proceed as in normal PCR except with this modified reaction composition (for a 50μl reaction) and cycle settings:

Name 50 μl reaction Final concentration
Water (reverse osmosis) 35.7 μl
10mM dNTPs 1 μl 200 μM each
10× NH4 buffer 5 μl 1×
Forward Primer 10 μM 2.5 μl 0.5 μM
Reverse Primer 10 μM 2.5 μl 0.5 μM
Template cells 1.3 μl liquid culture or a picked colony
Taq 5u/μl 1 μl 0.1 u/μl

Settings of PCR machine:

Step 1 (cell breakage) 95°C 6 min
Step 2 (cycle) 98°C

55°C

72°C

10 s

30 s

180 s

Step 3 (final extension) 72°C 5 min

Safety

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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