DNA handling

2.3.1 PCR

The Polymerase Chain Reaction (PCR) was used to amplify RYR1 cDNA fragments using first strand cDNA as a template. Reaction cycles were performed using an FTS-320 thermal sequencer (Corbett Research) or GeneAmp® PCR System 2700 (Applied Biosystems). Platinum® Pfx DNA Polymerase (Invitrogen) was used in this project for cloning and site-directed mutagenesis. This enzyme has 3’ to 5’ exonuclease proofreading activity and thus provides higher fidelity than Taq polymerase. Reaction composition and cycles were designed as general procedures according to the manufacturer’s instructions.

2.3.2 Site-directed mutagenesis

Site-directed mutagenesis for mutant RYR1 cDNA construction was performed with megaprimer PCR using whole plasmids (MEGAWHOP) [Miyazaki and Takenouchi, 2002]. This is a modified technique of the QuikChange® method amplifying initial megaprimers. PCR was performed using Platinum® Pfx DNA Polymerase and either

forward or reverse primers containing a mutation. PCR reactions for mutagenesis consisted of 1 cycle of 94°C for 5 minutes for denaturation followed by 20 cycles of 94°C for 30 seconds, 60°C for 30 seconds and 68°C for 30 seconds followed by 25 cycles of 94°C for 1 minute, 60°C for 1 minute and 68°C for 10 minutes followed by 1 cycle of 68°C for 5 minutes. Reaction mixtures were then diluted two fold with water and incubated with 1 µL of DpnI at 37°C for 1 hour.

2.3.3 DNA electrophoresis

DNA fragments were analysed by gel electrophoresis using 1% agarose LE, 0.4 µg/mL ethidium bromide and 1× TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) as the electrophoresis buffer. DNA samples were loaded onto gels with 5× DNA loading buffer (0.4% bromophenol blue, 0.1% xylene cyanol FF, 15% Ficoll in water). The gels were electrophoresed for 60 minutes at 80-100 V and DNA fragments were visualised under UV light.

2.3.4 DNA purification

DNA fragments needed to be purified for direct sequencing, endonuclease digestion or ligation. PCR fragments and DNA fragments after endonuclease digestion were purified using Wizard® SV Gel and PCR Clean-Up System (Promega) and plasmids constructed for RYR1 cloning were purified using Quantum Prep® Plasmid Miniprep Kit (Bio-Rad) or PureLink™ HiPure Plasmid Midiprep Kit (Invitrogen). DNA quantification was performed by measuring absorbance at 260 and 280 nm using an Ultraspec 3000 UV/Visible Spectrophotometer (Pharmacia Biotech) with 100 µL cuvettes. An absorbance at 260 nM of 1.0 is equivalent to a [DNA] of 50 µg/mL, while an A260/280 ratio of 1.8 is considered to be pure DNA [Sambrook, et al., 1989].

2.3.5 Direct sequencing

After subcloning followed by purification, 300-500 ng/μL of plasmid DNA was directly sequenced using an ABI 3730 Genetic Analyser with Big Dye terminator chemistry. Sequencing primers were diluted to 3.2 pmol/μL for use. This procedure was performed at the Allan Wilson Centre Genome Service by Ms Lorraine Berry

[Thompson, et al., 1994].

2.4 Cloning

The human RYR1 cDNA is 15,117 bp long and therefore it was not feasible to amplify the full length of this coding region from mRNA in one step. Approximately 1 kb fragments were amplified by PCR using the first strand cDNA as a template, and then these PCR products were subcloned in pBlueScript II KS or SK (+) (Stratagene) and were sequenced directly. Plasmids were then digested by restriction endonucleases for subsequent ligation and subcloning. The entire RYR1 cDNA was cloned in the mammalian expression vector, pcDNA3.1(+) (Invitrogen) for RYR1 protein expression in HEK-293 cells.

2.4.1 Restriction endonuclease digestion

Plasmids were digested with one or two restriction endonucleases to obtain RYR1 cDNA fragments. Conditions including buffer and temperature are dependent on the combination of restriction endonucleases although most reactions were performed overnight at 37-50°C in a total volume of 20 µL with 1 µg DNA, 100 µg/mL of bovine serum albumin (BSA) and 1 µL (~20 units) of restriction endonuclease. Reaction mixtures were electrophoresed and DNA fragments of interest were excised from the gel and purified using Wizard® SV Gel and PCR Clean-Up System (Promega) if the fragments were required for subsequent cloning.

2.4.2 Ligation

Ligation was performed using T4 DNA ligase (Invitrogen) according to the manufacturer’s instructions. Most reactions were performed in a total volume of 10 µL including 1 µL (1 unit/µL) of ligase, 2 µL of 5× ligase reaction buffer (250 mM Tris-HCl, pH 7.6, 50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) polyethylene

glycol-8000), digested plasmid vectors pBluescript or pcDNA3.1 (10-100 pg), and one or two digested DNA fragment inserts (~1 ng in total). Molar concentrations of inserts were normally 3-10 times higher than molar concentrations of the plasmid vectors. Samples were incubated at 4°C in fridge overnight for small inserts or 16°C overnight for long inserts.

2.4.3 Transformation of E. coli

Transformation was carried out using E. coli DH5α competent cells prepared by the Inoue method [Inoue, et al., 1990]. A single colony of DH5α was picked up and incubated in 25 mL of SOB (20 g of bacto tryptone, 5 g of bacto yeast extract, 0.5 g NaCl, 10 mL of 250 mM KCl, 0.2 mL of 5 M NaOH, 10 mL of 1 M MgCl2, 10 mL of 1

M MgSO4 and H2O to 1 L) at 37°C 6-8 hours at 250 rpm shaking. Ten mL of this

starter culture was added to 250 mL of SOB and incubated at 18-22°C overnight at 200 rpm shaking. After OD600 reached 0.4-0.8, cells were placed on ice. After pelleting,

cells were resuspended in 80 mL of transformation buffer (55 mM MnCl2, 15 mM

CaCl2, 250 mM KCl, 10 mM PIPES, pH6.7). Cells were pelleted again and

resuspended in 20 mL transformation buffer and 1.5 mL of DMSO was added. Cells were aliquoted in 100 µL and freezed immediately in liquid nitrogen. Frozen tubes were stored under -70°C.

Five µL of the ligation mixture was mixed with 100 µL of competent DH5α cells for 20 minutes on ice. Sample mixtures were incubated at 42°C for 1 minute and then plunged in ice for 2 minutes. Samples were diluted with 900 µL of SOC (20 g of bacto tryptone, 5 g of bacto yeast extract, 0.5 g NaCl, 0.2 mL of 5 M NaOH, 10 mL of 1 M MgCl2, 10 mL of 1 M MgSO4, 20 mL of 1 M glucose and H2O to 1 L) and were

incubated at 37°C for 1 hour. Fifty to 200 µL of these diluted mixtures were streaked on LB agar plates containing 50 µg/mL ampicillin with 50 µL of 20 mg/mL X-gal and 20 µL of 0.1 M IPTG spread on the surface. Plates were incubated at 37°C overnight but not over 16 hours.

2.4.4 Inoculation

Single colonies were picked into 500 µL of super LB (SLB, LB with 20 mM glucose and 20 mM MgCl2) for manual plasmid preparation, 2 mL of LB for miniprep or 50

mL of LB for midiprep. Solutions were incubated at 30-37°C for 12-16 hours with constant shaking. Inoculation using SLB was performed in duplicate to use one sample for the manual preparation and the other sample for preparation of glycerol stocks.

2.4.5 Manual plasmid preparation

After subcloning, manual plasmid preparations were performed initially to check that cloning was successful. Cells in 500 µL of SLB were pelleted by centrifugation. The other sample was mixed with 200 µL of 50% glycerol and stored at -80°C. Cell pellets were resuspended in 100 µL of Solution I (50 mM glucose, 25 mM Tris, 10 mM EDTA, pH 8.0) on ice. Two hundred µL of Solution II (0.2 M NaOH and 1% SDS) was added and incubated for 5 minutes on ice. One hundred and fifty µL of Solution III (3 M potassium acetate, 9.2 mL glacial acetic acid, H2O to 80 mL) was added and incubated

for 5 minutes on ice. After 5 minutes centrifugation at 12,000 g at 4°C, the supernatant was collected and mixed with 500 µL isopropanol. After 5-10 minutes, samples were centrifuged 10 minutes at 12,000 g at 4°C and DNA pellets were collected and then washed with 1 mL of 70% ethanol. DNA pellets were dried using a speedvac and redissolved in 30 µL of water and incubated at 37°C for 1 hour with 1 µL of RNase A (10 mg/mL) to dissolve the DNA completely and digest unwanted RNA. Yields were typically 100-130 ng/µL.

2.4.6 Mini or Midiprep

If the manual preparation showed that the subcloning was successful, cells from the same colony were used to inoculate 2 mL of LB and were incubated at 37°C overnight. A Quantum Prep® Plasmid Miniprep Kit (Bio-Rad) was used to purify plasmids of interest for restriction endonuclease digestion and further subcloning.

The full length WT or mutant RYR1 cDNA clone was used to inoculate 50 mL of LB from colonies for midiprep using PureLink™ HiPure Plasmid Midiprep Kit (Invitrogen). The incubation time after inoculation of RYR1 cDNA clone was strictly controlled. Over 12 hours of incubation at 37°C did not produce successful plasmid propagation in E.coli DH5α probably because the size of RYR1 is too large for E.coli to carry the plasmid for a long length of time. Incubation was performed first at 30°C overnight (12-16 hours) and then at 37°C for 1-5 hours depending on the cell density observed visually in the glass flask. Manual plasmid preparation was performed first using 1 mL of the 50 mL culture to check that the inoculation had been successful and the condition of the RYR1 cDNA clone was acceptable. Midipreps were performed

according to the manufacturer’s instructions. Yields of plasmid DNA were typically 1 µg/µL in 100 µL TE.