2 Materials
General laboratory reagents were purchased as analytical grade
from Sigma-Aldrich (USA), Millipore (UK), Fisher Scientific
(UK) and Thermo Fisher Scientific (UK). Other chemical reagents
and kits (all chemicals included) for the molecular biology were
obtained from Qiagen (Germany), Machinery-Nagel (USA), and
Agilent (UK) unless otherwise stated. All primers were purchased
from Integrated DNA Technologies (IDT, Europe) at a 25 nmole
(0.27 mg) DNA oligo concentration with standard desalting.
FuGENE HD Transfection reagent was purchased from Promega.
All cell culture reagents were bought from Gibco (Thermo Fisher
Scientific).
1. All primers were designed with a Tm (melting temperature)
close to 60
C, although this varied slightly depending on the
GC content. Gene-specific primers with 15 bp extension
homologous to vector ends were designed for In-Fusion™
cloning [28], using an automated primer design tool:
https://www.oppf.rc-harwell.ac.uk/Opiner/, or for fusing
two inserts together using SnapGene
® software (from GSL
Biotech; available at snapgene.com). Lyophilized primer stocks
(100 μM) were resuspended with nuclease-free water. Stocks
were diluted 1:10 to make up 10 μM working stocks that were
stored at À20
C.
2. Buffers and media for cloning the fluorescent protein tagged
proteins were prepared at room temperature using ultrapure
water (deionized water, at 18 MΩ-cm at 25
C). The S6K1GFPSpark construct was obtained from Sino Biological
(China) and mCherry-raptor and YFP-mTOR from Addgene.
Miniprep and Maxiprep kits were purchased from Qiagen
(UK).
3. HEK293 cell line was purchased from ATCC
® to make sure of
origin and purity and free from mycoplasma contamination as
well as passage number. Cell culture dishes with glass bottom
(35 mm) were bought from MatTek.
3 Methods
3.1 Amplification
of cDNA by PCR
PCRs were set up on ice in 0.2 ml PCR tubes or in an eight-strip
PCR tube (Star Lab) containing both forward (Fwd) and reverse
(Rev) primers at a 10 μM working concentration, Phusion Flash
Mix (Thermo Fisher Scientific), template plasmid DNA
(100–200 ng) containing cDNA gene to be amplified and sterile
water as shown in Table 1. Constituents were gently mixed by
292
Abdullah Ahmed et al.
General laboratory reagents were purchased as analytical grade
from Sigma-Aldrich (USA), Millipore (UK), Fisher Scientific
(UK) and Thermo Fisher Scientific (UK). Other chemical reagents
and kits (all chemicals included) for the molecular biology were
obtained from Qiagen (Germany), Machinery-Nagel (USA), and
Agilent (UK) unless otherwise stated. All primers were purchased
from Integrated DNA Technologies (IDT, Europe) at a 25 nmole
(0.27 mg) DNA oligo concentration with standard desalting.
FuGENE HD Transfection reagent was purchased from Promega.
All cell culture reagents were bought from Gibco (Thermo Fisher
Scientific).
1. All primers were designed with a Tm (melting temperature)
close to 60
C, although this varied slightly depending on the
GC content. Gene-specific primers with 15 bp extension
homologous to vector ends were designed for In-Fusion™
cloning [28], using an automated primer design tool:
https://www.oppf.rc-harwell.ac.uk/Opiner/, or for fusing
two inserts together using SnapGene
® software (from GSL
Biotech; available at snapgene.com). Lyophilized primer stocks
(100 μM) were resuspended with nuclease-free water. Stocks
were diluted 1:10 to make up 10 μM working stocks that were
stored at À20
C.
2. Buffers and media for cloning the fluorescent protein tagged
proteins were prepared at room temperature using ultrapure
water (deionized water, at 18 MΩ-cm at 25
C). The S6K1GFPSpark construct was obtained from Sino Biological
(China) and mCherry-raptor and YFP-mTOR from Addgene.
Miniprep and Maxiprep kits were purchased from Qiagen
(UK).
3. HEK293 cell line was purchased from ATCC
® to make sure of
origin and purity and free from mycoplasma contamination as
well as passage number. Cell culture dishes with glass bottom
(35 mm) were bought from MatTek.
3 Methods
3.1 Amplification
of cDNA by PCR
PCRs were set up on ice in 0.2 ml PCR tubes or in an eight-strip
PCR tube (Star Lab) containing both forward (Fwd) and reverse
(Rev) primers at a 10 μM working concentration, Phusion Flash
Mix (Thermo Fisher Scientific), template plasmid DNA
(100–200 ng) containing cDNA gene to be amplified and sterile
water as shown in Table 1. Constituents were gently mixed by
292
Abdullah Ahmed et al.