275
7. Commercial DNA plasmid purification kit (e.g., QIAprep Spin
Miniprep Kit).
8. Commercial yeast transformation kit: e.g., the Frozen-EZ
yeast transformation II kit (Zymo Research).
Primer characteristics for each application:
1. Overlap extension PCR (OE-PCR): presence of G or C at 5′
end; T m between 64 and 66 °C; the overlapping region with
the target sequence has to be 15–22 bp long.
2. Terminal Extension PCR (TE-PCR): 5′ extension can be up to
35 bp long; T m of region annealing to target sequence has to
be between 64 and 66 °C.
3. Site directed mutagenesis PCR (SDM-PCR): primer length
should be between 42 and 45 bp; the desired mutation has
to be close to the center of the primer sequence; the T m
(calculated as per QuikChange II XL Site-Directed Mutagenesis
Kit) has to be greater than 78 °C.
4. Error-prone PCR (EP-PCR): the primer binding sites are
located on the vector sequence at a distance greater than 70 bp
from desired restriction sites. T m is between 64 and 66 °C. In
Error-Prone PCR reactions, the required primers stocks concentration is 250 ng/μL.
1. Functionally effective Blue light: Tri-Star Rebel LED (Luxeon
#MR-R0500-20T) containing three Royal-Blue LEDs, mounted
on 20 mm Tri-Star Saber base: 2730 mW at recommended
operating current 700 mA; λ = 447.5 nm (440–460 nm); beam
angle 125°.
2. LED Heat Sink: Alpha Heat Sink (Luxeon #N80-20B) with
convection thermal resistance rating of 2.85 °C/W and
80 × 80 × 20 mm footprint.
3. LED adhesive tape: thermally conductive, electrically isolating
and adhesive Bond-Ply 100 pad (Luxeon #LXT-T-12) cut to
fit the 20 mm Luxeon LED’s Tri-Star base. Operational temperature from −30 °C to 120 °C; thermal performance
4.5 °C/W.
4. Functionally ineffective Red light: Tri-Start Rebel LED
(Luxeon #MR-H2060-20T) containing three Red–Orange
LEDs, mounted on 20 mm Tri-Star Saber base: 366 mW at
recommended operating current 700 mA; λ = 617 nm
(610–620 nm); beam angle 125°.
5. Safety light: Deep-Orange co-extruded polycarbonate film
(Rosco Supergel #22) applied to main room light source: RGB
240:80:0; transmittance ~0% at λ <540 nm.
2.6.2 PCR Primers
2.7 Light Irradiation
Engineering Light-Regulated K
+
Channels
7. Commercial DNA plasmid purification kit (e.g., QIAprep Spin
Miniprep Kit).
8. Commercial yeast transformation kit: e.g., the Frozen-EZ
yeast transformation II kit (Zymo Research).
Primer characteristics for each application:
1. Overlap extension PCR (OE-PCR): presence of G or C at 5′
end; T m between 64 and 66 °C; the overlapping region with
the target sequence has to be 15–22 bp long.
2. Terminal Extension PCR (TE-PCR): 5′ extension can be up to
35 bp long; T m of region annealing to target sequence has to
be between 64 and 66 °C.
3. Site directed mutagenesis PCR (SDM-PCR): primer length
should be between 42 and 45 bp; the desired mutation has
to be close to the center of the primer sequence; the T m
(calculated as per QuikChange II XL Site-Directed Mutagenesis
Kit) has to be greater than 78 °C.
4. Error-prone PCR (EP-PCR): the primer binding sites are
located on the vector sequence at a distance greater than 70 bp
from desired restriction sites. T m is between 64 and 66 °C. In
Error-Prone PCR reactions, the required primers stocks concentration is 250 ng/μL.
1. Functionally effective Blue light: Tri-Star Rebel LED (Luxeon
#MR-R0500-20T) containing three Royal-Blue LEDs, mounted
on 20 mm Tri-Star Saber base: 2730 mW at recommended
operating current 700 mA; λ = 447.5 nm (440–460 nm); beam
angle 125°.
2. LED Heat Sink: Alpha Heat Sink (Luxeon #N80-20B) with
convection thermal resistance rating of 2.85 °C/W and
80 × 80 × 20 mm footprint.
3. LED adhesive tape: thermally conductive, electrically isolating
and adhesive Bond-Ply 100 pad (Luxeon #LXT-T-12) cut to
fit the 20 mm Luxeon LED’s Tri-Star base. Operational temperature from −30 °C to 120 °C; thermal performance
4.5 °C/W.
4. Functionally ineffective Red light: Tri-Start Rebel LED
(Luxeon #MR-H2060-20T) containing three Red–Orange
LEDs, mounted on 20 mm Tri-Star Saber base: 366 mW at
recommended operating current 700 mA; λ = 617 nm
(610–620 nm); beam angle 125°.
5. Safety light: Deep-Orange co-extruded polycarbonate film
(Rosco Supergel #22) applied to main room light source: RGB
240:80:0; transmittance ~0% at λ <540 nm.
2.6.2 PCR Primers
2.7 Light Irradiation
Engineering Light-Regulated K
+
Channels