Single Colony PCR

Colony PCR
Colony PCR is useful when you are doing genomic mutations but don’t want to take the day to isolate the genome. It has worked well with Pfx DNA Polymerase.

1. Pick colonies. Specifically, pick colonies with a pipette tip and resuspend in 20 μl of cold ddH₂O by pipetting up and down
   Pipette 3μl onto an index plate with appropriate antibiotic for use later if colony is good.

• CAREFUL! Do if you go to the second plunge with the pipette tip, it will spatter and you can get contaminant cells!

1. Grow index plate at 37°C o/n.
2. Make master mix:…………..20 μl/rxn
3. ## 10x PCR buffer……….5.0
4. ## 10mM dNTPs…………0.6
5. ## 50mM Mg₂SO₄……….0.4
6. ## 10x enhancer…………6.0
7. ## ddH₂O…………………3.0
8. #*”Note: mix together {n+1} volumes of each substrate, where n=the number of reactions you will be doing.
9. #*”Note: these volumes are for 20uL reactions. Adjust if using lower volumes.”
10. # Make 10μM primer mix:
11. ## Mix 2μL of both primers (100μM stock) into 18μL ddH₂O.
12. ## If you need more than 20μL of primer, adjust volumes. (1μL of each per total 10μL mix)
13. # For each 20 μl reaction, mix together in PCR tube.

• 15μL Master Mix
• 2.0μL Colony suspension (template)
• 2.0μL Primer mix (10μM each primer)
• 1.0μL Pfx Platinum DNA Polymerase

1. Program cycle in PCR thermocycler with steps 2-4 repeating 34 times.

• 94°C at 5:00 (m:s)
• 94°C at 0:15
• 55°C at 0:30
• 68°C at 2:00
• 68°C at 7:00
• 4°C at ∞

1. Check products on a gel with 10μL samples (2.5μL 5xdye). Should be the same size as the PCR product from earlier. Also–run a control using the host strain with pKD46. This should result in the length of the gene(s) to be knocked out+100.