PCR

Primestar Max PCR protocol — for plasmid

1. General Composition of PCR Reaction Mixture

NOTE: When using plasmids as the template, the mass of the mixture should not exceed 1ng.

2. PCR condition

Before cycles: 98°C 10 After cycles: 72°C 7min; 16°C ∞ (4°C is more suitable for storing PCR product, but storing 4°C overnight may result in damage of some PCR equipments)

(A) For reactions in which the quantity of template is 200 ng / 50 μl or less:*
-98°C 10 sec.
-55°C 5 sec. or 15 sec
-72°C 5 sec./kb
-30-35 cycles

(B) For reactions in which the quantity of template exceeds 200 ng / 50 μl:*
-98°C 10 sec.
-55°C 5 sec. or 15 sec.
-72°C 30 - 60 sec./kb
-30-35 cycles

OR
-98°C 10 sec.
-68°C 30 - 60 sec./kb
-30 - 35 cycles [2-step PCR]

NOTE: For amplifying fragments more than 3kb, extension time should be increased to 40 sec./kb

・Denaturing conditions: 98°C for 5 to 10 sec. is recommended. If performing denaturation at 94°C, set the denaturation step for 10 to 15 sec.

・Annealing temperature: Use 55°C as the default annealing temperature.

Annealing time:

For primers that are 25-mer or shorter:

-For primer Tm values (calculated by the formula below) of 55°C or greater, anneal for 5 sec.
-For primer Tm values (calculated by the formula below) less than 55°C, anneal for 15 sec.

For primers longer than 25-mers:

-Use an annealing time of 5 sec.
* Tm value calculation method:
Tm (°C) = 2(NA + NT) + 4(NC + NG) - 5
where N represents the number of primer nucleotides having the specified identity (A, T, C, or G)

TaKaRa Premix Taq PCR protocol — for bacterial colony

1. General reaction mixture for PCR

Premix Taq    25ul
Template    <500ng
Primer 1      0.1-1.0uM(final conc.)
Primer 2    0.1-1.0uM(final conc.)
Sterilized distilled water     up to 50ul

Safety

Out of safety concerns, instead of using the pathogenic Fusobacterium nucleatum, we transform a section of FadA’s coding sequence — with sgRNA’s binding site — into E.coli to test our system’s efficiency.