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Materials
You will need:
- 10X Buffer
- Q solution
- Forward primer
- Reverse primer
- dNTP
- TAQ Polymerase
- Sterile milliQ water
Extra Notes
None.
Procedure
Making the Cocktail
- Make a little more cocktail than is actually required to account for pipetting accuracy. Eg. if 10 reactions are to be done, multiply the volume of each ingredient by 10.5. The following table indicates how much cocktail is needed for 1 rxn.
| Ingredient |
μL/rxn (20μL/rxn) |
μL/rxn (50μL/rxn) |
| 10X Buffer |
2 |
5 |
| Q solution |
4 |
10 |
| Forward primer |
1 |
2.5 |
| Reverse primer |
1 |
2.5 |
| dNTP |
0.5 |
1.25 |
| TAQ Polymerase |
0.125 |
0.3 |
| Sterile milliQ water |
1.375 |
3.5625 |
- In each PCR tube that you plan to perform a PCR reaction in, put 10μL of sterile milliQ water, pluck your desired colony with a 20μL pipette tip (using 2x microscope if necessary) and place tip in PCR tube. (If you are using a DNA sample in solution as your template, than alter volume of water in PCR tube; eg. add 2μL template DNA to 8μL milliQ water in PCR tube.)
- Add 10μL/40μL depending in reaction volume) to respective PCR tube.
- Spin down PCR tubes in picocentrifuge.
- Program thermal cycler to perform steps:
| Step number |
Step Description |
Temperature (°C) |
Time (minutes) |
| 1 |
Initial Denaturation |
98 |
10:00 |
| 2 |
Denaturation |
98 |
0:45 |
| 3 |
Primer Annealing |
55 |
1:00 |
| 4 |
Fragment Extension |
72 |
1 minute PER kb of PCR product |
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Go to Step #2 25 more times |
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| 5 |
Extending incomplete fragments |
72 |
5:00 |
Purpose
To screen the colonies that have the desired parts.
References
- Written by D. Larsen from notes given by M. Facciotti
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We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!
We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)
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