Difference between revisions of "Team:BostonU/Protocols"
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<th>Extension Time</th> | <th>Extension Time</th> | ||
| − | <td>If target size is: <BR>< 500 bp run 10 sec/kb<BR>500-1000 bp run 15 sec/kb<BR>1000-3000 bp run 20 sec/kb<BR>< 3000 bp run 20 sec/ | + | <td>If target size is: <BR>< 500 bp run 10 sec/kb<BR>500-1000 bp run 15 sec/kb<BR>1000-3000 bp run 20 sec/kb<BR>< 3000 bp run 20 sec/kb</td> |
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</table> | </table> | ||
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<th>Extension Time</th> | <th>Extension Time</th> | ||
| − | <td>If target size is: <BR>< 500 bp run 10 sec/kb<BR>500-1000 bp run 15 sec/kb<BR>1000-3000 bp run 20 sec/kb<BR>< 3000 bp run 20 sec/ | + | <td>If target size is: <BR>< 500 bp run 10 sec/kb<BR>500-1000 bp run 15 sec/kb<BR>1000-3000 bp run 20 sec/kb<BR>< 3000 bp run 20 sec/kb</td> |
</tr> | </tr> | ||
</table> | </table> | ||
Revision as of 16:06, 28 October 2017
OUR TEAM
Below is an interactive list of the laboratory techniques employed by the team. Click to expand for details.
Colony PCR
Materials
- 5% DMSO
- 2x KOD Hot Start Master Mix
- PCR Tubes
- Forward Primers(s) [10 uM]
- Reverse Primer(s) [10 uM]
- DNA Template
- Deionized water
Procedure
- Add 18.5 uL of deionized water to PCR tube
- Add 1.5 µl of both the requisite Forward and Reverse Primers to PCR tube
- Add 1 ul of DNA template
- Add 27.5 µl of KOD Hot start Master Mix to PCR tube
- Centrifuge for 10 seconds to remove air bubbles
- Place in Thermocycler
Thermocycler Conditions
| Number of Cycles | From 20 - 40 |
|---|---|
| Annealing Temperature | Set temperature to the lowest primer melt temperature |
| Extension Time | If target size is: < 500 bp run 10 sec/kb 500-1000 bp run 15 sec/kb 1000-3000 bp run 20 sec/kb < 3000 bp run 20 sec/kb |
INSERT
Materials
- 5% DMSO
- 2x KOD Hot Start Master Mix
- PCR Tubes
- Forward Primers(s) [10 uM]
- Reverse Primer(s) [10 uM]
- DNA Template
- Deionized water
Procedure
- Prepare the DNA template by picking a colony from a plate and mixing it into 10 uL of deionized water in a PCR tube
- Add 17.5 µl of deionized water to another PCR tube
- Add 1.5 µl of both the requisite Forward and Reverse Primers to the second PCR tube
- Add 2 ul of water containing cells with DNA template sample
- Add 27.5 µl of KOD Hot Start Master Mix
- Centrifuge for 10 seconds to remove air bubbles
- Place in Thermocycler
Thermocycler Conditions
| Number of Cycles | From 20 - 40 |
|---|---|
| Annealing Temperature | Set temperature to the lowest primer melt temperature |
| Extension Time | If target size is: < 500 bp run 10 sec/kb 500-1000 bp run 15 sec/kb 1000-3000 bp run 20 sec/kb < 3000 bp run 20 sec/kb |
