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| - | ==Colony PCR:==
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| - | 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.
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| - | {| class="wikitable" style="text-align: center; color: purple;"
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| - | ||Reagent||Volume (µl)||Final Concentration
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| - | ||Water||35.7||
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| - | ||10 mM dNTPs||1||200 µM
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| - | ||10 x NH4 buffer||5||1x
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| - | ||Forward Primer||2.5||0.5 µM
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| - | ||Reverse Primer||2.5||0.5 µM
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| - | ||Template Cells||1.3 (from liquid culture or picked colony||
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| - | ||Taq polymerase 5u/µl||1||0.1 u/ µl
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| - | |}
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| - | PCR machine settings:
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| - | {|class="wikitable" style="text-align: center; color: purple;"
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| - | |colspan="2"| ||Temperature (oc)||Time (s)
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| - | |colspan="2"|Step 1 (cell breakage)||95||360
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| - | |rowspan="3"|Step 2 (Cycle 30x)||Denaturing||98||10
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| - | ||Annealing||60||30
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| - | ||Elongation||72||120
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| - | |colspan="2"|Step 3 (final extension)||72||300
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| - | |}
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| - | <center>[[Team:Cambridge/Protocols|'''Back to Protocols''']]</center>
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| - | <center>[[Team:Cambridge/Notebook|'''Back to the Notebook''']]</center>
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Latest revision as of 15:22, 13 July 2012
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