Economic and easy bacterial and yeast colony PCR with 2x premix Gotaq-Green (Promega) or DreamTaq-Green (Thermo Fisher)
Dariusz Abramczyk
Published: 2023-03-18 DOI: 10.17504/protocols.io.eq2ly74jqlx9/v1
Abstract
Steps
1.
Bacterial colony PCR
PCR reactions using ProFlex PCR system, Applied Biosystems. Thermal Cycler
1.1.
Maximum (good quality) PCR product up to ~3kb. The best yield is 0.2kb-2kb
pick a colony from agar plate and swirl in sterile water 50-100µL . From liquid culture, dilute saturated culture5-10µL in sterile 50-100µL water.This is a source for reaction (taking 0.8µL ).Keep the rest in ice, for re-culture of positively verified clones.
| 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
Note
1.2.
Load on 1-1.2% agarose gel 8-10µL and visualise under GelDoc or another UV source.
2.
Yeast colony PCR (S.cerevisiae, Pichia, Candida)
2.1.
Alkaline yeast cells lysis treatment. Pick a yeast colony from agar plate and swirl in sterile 20mM NaOH 50-100µL .
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 0h 10m 0s and immediately transfer to ice/4°C for at least 0h 5m 0s
2.2.
Maximum PCR product up to ~3kb. The best yield is 0.2kb-2kb
Important - annealing 1min30sec
| 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
Note
2.3.
Load on 1-1.2% agarose gel 8-10µL and visualise under GelDoc or another UV source.
