Colony PCR
Updated 9/27/2021 07:05 PM
Introduction
Materials
Procedure
- Make master plates by dividing each LB agar plate in 6 numbered sections, continue numbering on the next plate.
- Thaw solutions for PCR master mix on ice.
- Suspend primers to concentration 100µM (see specification sheet)
- Gently vortex and briefly centrifuge all solutions after thawing.
- Make dNTP Mix by adding equal volumes of each dNTP (100mM) to a sterile tube.
- Make a master mix: enough for wanted number of 20µL reactions + 1 (for margins)
a) Number of 20µL reactions: 1 per clone + 1 negative (no template DNA)
b) Recipe for 20µL reaction, mix thoroughly:
Table1
A A | B B | |
1 1 | Solution | µL |
2 2 | 10x Taq Buffer with KCl | 2 |
3 3 | Each dNTP (100mM each) | 0.04 OF EACH (0.2 mM final conc of each) |
4 4 | Forward primer (100 µM) | 0.06 (0.3 µM final conc) |
5 5 | Reverse primer (100 µM) | 0.06 (0.3 µM final conc) |
6 6 | MgCl2 (25 mM) | 1.2 (1.5 mM final conc) |
7 7 | Taq DNA Polymerase (5 U/µL) | 0.1 (0.5 U) |
8 8 | Nuclease free water | 16.42 (to reach 20 µL) |
- Place thin-walled PCR tubes on ice and add 20µL master mix to each
- Pick a single colony (clone) from a transformant plate with a sterile inoculation loop and streak on the center of an empty (but labelled) section of a master plate. Rinse the same loop in a PCR tube with master mix (20µL reaction).
a) Repeat for each clone, ~3 clones per successful transformant plate. Remember to take from the plate with intact plasmid without insert.
b) Incubate master plates at 37°C until step 12.
- Gently vortex the PCR tubes and spin down
- Perform PCR:
Table2
A A | B B | C C | D D | |
1 1 | Step | Duration | Temperature | Number of cycles |
2 2 | Pre-heating | 10 min | 95 °C | 1 |
3 3 | Denature | 30 sec | 95 °C | 30 |
4 4 | Annealing | 30 sec | 61 - 5 °C (Depends on primers)=56°C | 30 |
5 5 | Elongation | 1 min/kbp | 72 °C | 30 |
6 6 | Final elongation | 10 min | 72 °C | 1 |
- Analyse on gel to detect presence of PCR product of expected length.
- For each clone which yielded the correct PCR product: inoculate overnight culture using the colony from the correct section of the master plate after it has had time to incubate.
- Store master plates in the fridge or cold room.
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