Oct 18, 2020
Plaque PCR
- 1South China University of Technology
Protocol Citation: Jiaxin Li 2020. Plaque PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bni9mch6
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: October 18, 2020
Last Modified: October 18, 2020
Protocol Integer ID: 43329
Mark independent plaques on the back of the Petri dish with a black pen. Take 6 points in a plate and try to take points randomly and evenly.
Take 24 1.5 ml EP tubes.
A) Add 200 μl SM Buffer per tube
B) Add 2 μl chloroform to each tube
Sampling
Sampling
Suck out the plaque just spotted with the pipette
Put into the prepared EP tube.
Open the metal bath to 95 degrees for lysis of the phage
Place the EP tube at room temperature for one hour to dissolve the sample sufficiently and mix several times during the period.
PCR system
PCR system
KOD FX 20ul system
Add 18.4 μl to each tube (1.6 μl template is needed)
After an hour, put the EP tube in a metal bath and heat it for 10 minutes.
Centrifugation for 5 min, 12,000 rpm
Take 1.6ul supernatant from EP tube and add it to new EP tube.
Centrifuge the EP tube briefly with a centrifuge.
Place in PCR instrument.