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
Mechanism of FadA protein
The FadA protein is activated when its two forms combine and become internalized. The first form is a pre-FadA that is anchored in the cell membrane, whereas the second form is the mature FadA (mFadA) that is secreted out of F. nucleatum. When the two forms combine to form a complex, the protein is capable to help F. nucleatum bind to the host epithelial cell, thus allowing F. nucleatum embark on invading the host cells.
Overview (Fig. 1)
We will first express Cas9 and sgRNA in E.coli and then transport them to E.coli’s periplasm(Step 1). By then, they may be packaged by OMVs that bud off from E.coli’s outer membrane(Step 2). Those OMVs will be collected and mixed with our target bacteria, thereby allowing them to fuse with bacteria again, to release the Cas9 proteins and sgRNA(Step 3), and cleave the target gene (Step 4 & 5). Afterwards, we will test whether the target gene is cleaved by Cas9.
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.
