Mar 17, 2023
Economic and easy bacterial and yeast colony PCR with 2x premix Gotaq-Green (Promega) or DreamTaq-Green (Thermo Fisher)
- Dariusz Abramczyk1
- 1University of Edinburgh
Protocol Citation: Dariusz Abramczyk 2023. Economic and easy bacterial and yeast colony PCR with 2x premix Gotaq-Green (Promega) or DreamTaq-Green (Thermo Fisher) . protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly74jqlx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 10, 2023
Last Modified: March 17, 2023
Protocol Integer ID: 78495
Keywords: yeast colony pcr, colony pcr, yeast, pcr, method gotaq promega, modified method gotaq promega, thermo fisher, promega, thermo
Bacterial colony PCR
GoTaq Green Master MixPromegaCatalog #M7122
PCR reactions using ProFlex PCR system, Applied Biosystems. Thermal Cycler
| A | B | C | |
| Reagent Name | Initial Conc. | Volume (μL) | |
| gotaq green 2x | 2X | 5 | |
| oligo 1 | 10uM | 0.6 | |
| oligo 2 | 10 uM | 0.6 | |
| cell suspension in water | cells suspension | 0.8 | |
| ddH2O | N/A | 3 | |
| TOTAL Volume: | 10 uL | ||
| A | B | C | D | |
| Number of Cycles | Step Name | Temperature | Duration | |
| 1 | Initial Denaturation | 98oC | 10min | |
| 30 | Denaturation | 98oC | 10 sec | |
| Annealing | 50-55oC | 30 sec | ||
| 1min (depends on the length) | 72oC | 1min per 1kb | ||
| 1 | Hold | 4-10oC | hold |
Note
Maximum (good quality) PCR product up to ~3kb. The best yield is 0.2kb-2kb
Note
pick a colony from agar plate and swirl in sterile water 50-100 µL . From liquid culture, dilute saturated culture5-10 µL in sterile 50-100 µL water.
This is a source for reaction (taking 0.8 µL ).
Keep the rest in ice, for re-culture of positively verified clones.
Load on 1-1.2% agarose gel 8-10 µL and visualise under GelDoc or another UV source.
Yeast colony PCR (S.cerevisiae, Pichia, Candida)
Alkaline yeast cells lysis treatment. Pick a yeast colony from agar plate and swirl in sterile 20mM NaOH 50-100 µL .
Optionally, a liquid culture or a agar-plate pick colony resusupend in the sterile water or YPD, take small volume (~20uL) and mix with the same volume of 40mM NaOH.
20mM NaOH-cells resuspension incubate 95 °C 00:10:00 and immediately transfer to ice/4 °C for at least 00:05:00
15m
| A | B | C | |
| Reagent Name | Initial Conc. | Volume (μL) | |
| gotaq green 2x | 2X | 5 | |
| oligo 1 | 10uM | 0.6 | |
| oligo 2 | 10 uM | 0.6 | |
| cell suspension in 20mM | yeast suspension | 0.8 | |
| ddH2O | N/A | 3 | |
| TOTAL Volume: | 10 uL | ||
| A | B | C | D | |
| A | B | C | D | |
| Number of Cycles | Step Name | Temperature | Duration | |
| 1 | Initial Denaturation | 98oC | 2min | |
| 30-33 | Denaturation | 98oC | 10 sec | |
| Annealing | 52-55oC | 1min 30sec | ||
| 1min (depends on the length) | 72oC | 1min per 1kb | ||
| 1 | Hold | 4-10oC | hold |
Note
Maximum PCR product up to ~3kb. The best yield is 0.2kb-2kb
Note
Important - annealing 1min30sec
Load on 1-1.2% agarose gel 8-10 µL and visualise under GelDoc or another UV source.