|
|
| Line 1: |
Line 1: |
| | | | |
| - | {| style="color:#1b2c8a;background-color:#3ae2e8;" cellpadding="3" cellspacing="0" border="1" bordercolor="#fff" width="62%" align="center"
| |
| - | !align="center"|[[Team:Cambridge|Home]]
| |
| - | !align="center"|[[Team:Cambridge/Team|Team]]
| |
| - | !align="center"|[https://igem.org/Team.cgi?year=2012&team_name=Cambridge Official Team Profile]
| |
| - | !align="center"|[[Team:Cambridge/Project|Project]]
| |
| - | !align="center"|[[Team:Cambridge/Parts|Parts Submitted to the Registry]]
| |
| - | !align="center"|[[Team:Cambridge/Modeling|Modeling]]
| |
| - | !align="center"|[[Team:Cambridge/Notebook|Notebook]]
| |
| - | !align="center"|[[Team:Cambridge/Safety|Safety]]
| |
| - | !align="center"|[[Team:Cambridge/Attributions|Attributions]]
| |
| - | !align="center"|[[Team:Cambridge/Sponsors|Sponsors]]
| |
| - | |}
| |
| - |
| |
| - | ==Colony PCR:==
| |
| - |
| |
| - |
| |
| - | <big>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.</big>
| |
| - |
| |
| - | {| class="wikitable" style="text-align: center; color: purple;"
| |
| - | ||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:
| |
| - |
| |
| - | {|class="wikitable" style="text-align: center; color: purple;"
| |
| - | |colspan="2"| ||Temperature (oc)||Time (s)
| |
| - | |-
| |
| - | |colspan="2"|Step 1 (cell breakage)||95||360
| |
| - | |-
| |
| - | |rowspan="3"|Step 2 (Cycle 30x)||Denaturing||98||10
| |
| - | |-
| |
| - | ||Annealing||60||30
| |
| - | |-
| |
| - | ||Elongation||72||120
| |
| - | |-
| |
| - | |colspan="2"|Step 3 (final extension)||72||300
| |
| - | |}
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <big>'''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.</big>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <center>'''[[Team:Cambridge/Protocols|Back to Protocols]]'''</center>
| |
"
