E coli colony PCR
- Prepare mastermix
- Divide 14μl mix / sample over PCR reaction tubes/wells
- Prepare cells in water
- Add 1 µl cell dilution to the mix
The E. coli colonies that grow on plates can be directly used as template. However, because too many cells can inhibit the PCR reaction, it is preferred to dilute these first in water and only use 1µl of this dilution. The success rate increases to close to 100%.
To dilute the colony:
- Mark the cells on the plate so you know which colonies you used.
- Fill a clean PCR strip or 96well plate with 50µl sterile water (does not have to be PCR grade, you'll be adding live bacteria anyways :)
- For each colony, touch the colony with a sterile toothpick.
- Place the toothpick in a tube/well with water, turn a few times and remove.
- Use 1μl of this mix to each reaction.
|Do sequencing reaction:|
|Taq PCR Buffer 10X||1.5 μl|
|dNTPs (10 mM each)||0.6 μl|
|MgCl2 (50mM)||0.6 μl|
|Primer Fwd (10 μM)||0.3 μl|
|Primer Rev (10 μM)||0.3 μl|
|Taq polymerase (5U/μl)||0.16 μl|
Run using program (bart -> colony PCR or cPCR)
|Number of cycles||Temperature||Duration|
|1 X||94°C||2 min|
|72°C||1 min 30 sec (or ~1kb/min)|