General molecular methods

2.2.1 Preparation of genomic DNA

Genomic DNA was extracted from leaf material with a method (Edwards, Johnstone and Thompson 1991). High-throughput genotyping was performed in 96 well blocks. For this, the plant tissue was ground with the Qiagen Mixer Mill. The DNA was isolated from leaf tissue by adding extraction buffer (100mM Tris pH 7.5, 50mM EDTA, 500mM NaCl, 10mM β-mercaptoethanol) and vortexing the sample for 5s. Afterwards SDS was added to samples to 0.5%. The extracts were centrifuged for 1 minutes and 300µl of supernatant was transferred to a fresh 96well block.

300µl of isopropanol was added to the mixture and precipitated at -20ºC for 1 hour. Following centrifugation for 10 minutes the supernatant was removed and the pellet washed with 70% ethanol, air dried for 1hour and re-suspended by pipetting in 100μl TE buffer.

2.2.2 PCR methods

Standard PCR reactions were set up in 40μl volumes containing 1.25 units of DNA Taq Polymerase (Invitrogen®), 4μl (10pg – 1µg) of DNA template, 0.1 – 1µM of gene specific forward and reverse primers, 0.2mM of each dNTP, 1-4mM of MgCl2. Standard PCR reactions were carried out with an initial denaturation step of

94°C for 1 minutes followed by cycles of denaturation at 94°C for 30s, annealing at a temperature deemed appropriate for the primer pair for 30s and extension at 72°C for 1 minutes per kb of product.

2.2.3 Genotyping of bacterial (E.coli) colonies

The presence of the desired fragment in the expression vector was confirmed by PCR using gene specific primers or in case of pGEM-T Easy M13For and M13Rev primers (Appendix 1). Toothpicks containing single colonies were placed in 5µl of water, and the PCR reagents were added up to the reaction volume of 40µl (1.25units of DNA Taq Polymerase (Invitrogen®), 0.1 – 1µM of gene specific forward and reverse primers, 0.2mM of each dNTP, 1-4mM of MgCl2). PCR

reactions were carried out with an initial denaturation step of 94°C for 5 minutes followed by 30 cycles of denaturation at 94°C for 30s, annealing at a temperature deemed appropriate for the primer pair for 30s and extension at 72°C for 1 minutes per kb of product.

2.2.4 Genotyping of LRR RLK mutants and T-DNA lines

A list of oligonucleotide sequences used to genotype the mutants and T-DNA lines are available in Appendix 1. Standard PCR conditions were used for genotyping plants unless stated otherwise in the relevant results section.

2.2.5 Agarose gel electrophoresis

Following PCR, agarose gel electrophoresis was carried out to visualise products. For a standard 40μl reaction, 3μl of Orange G (C16H10N2O7S2Na2, Sigma-Aldrich®, Cat. No. O3756) was added prior to loading. The size of products was estimated by loading 5μl (0.7μg/lane) of 1 kb Plus DNA ladder (Invitrogen Ltd., Cat. No. 10787). The separation of PCR products was visualized on gels made up using 1 x TAE buffer (VWR International, Cat. No. 44125D) containing either 1% or 1.5 % agarose and stained with ethidium bromide (final concentration 0.5µg/ml). Gels were run in tanks containing 1 x TAE buffer at approximately 100 mA (300V) for 20-60 minutes depending on the size of the products. Photographs were taken using a G:BOX gel documentation system (Syngene) and the images were analyzed using Gene Tools software.

2.2.6 Quantification of DNA

DNA concentrations were measured using a Nano-Drop spectrophotometer (Thermo Scientific). The measuring point was cleaned with Millipore water and soft tissue. The instrument was blanked using 1µl of distilled water on the measuring

point (blank measurement). To determine the concentration of the DNA, 1µl sample was loaded and the concentration was read on the PC monitor.

2.2.7 Preparation of cDNA from total RNA extracted from young seeds Hot borate extraction buffer was made up of 200mM sodium borate decahydrate (Borax) (pH 9), 30mM ethylene glycol bis (beta-aminoethyl ether)- N,N’-tetraacetic acid (EGTA), 1% (w/v) sodium dodecyl sulphate (SDS), 1% (w/v) sodium deoxycholate, 2% (w/v) polyvinylpyrrolidone (PVP) (Mr 40,000), 0.5% (v/v) Nonidet-40 (NP-40), and 10mM dithiothreitol (DTT) added just before grinding. The buffer was made with 0.1% DEPC (diethyl pyrocarbonate)–treated water and then autoclaved.

Approximately 100mg of frozen Arabidopsis early developed seeds dissected out of the siliques were homogenized in 1.5ml microcentrifuge tubes containing glass beads using a Simal S6 shaker. 1ml of 80°C heated extraction buffer and 60μl of Proteinase K (Progen Industries Limited, 25 mg/mL in H2O) was then added to

the frozen homogenized tissue and mixed by vortexing. The sample was transferred to a fresh 15ml tube and another 1ml of extraction buffer was added to a grinding tube to wash the remaining tissue. The tissue-buffer mix was loaded on two Qiashredder spin columns and microcentrifuged for 1minutes at 13000 rpm. The flow through was transferred to a fresh sterile tube and the loading and centrifugation step was repeated until the whole tissue was processed. 0.5 volume of absolute ethanol was added to the collected flow through and mixed by inverting several times. The sample was loaded on two Qiagen RNeasy mini columns and centrifuged for 1minutes at 13000rpm and the flow through discarded. An on- column DNase digestion was performed with the RNAase-Free DNase Set (Qiagen).

was followed. Samples were re-suspended in 40μl of RNase-free water and stored at -80°C. The first strand cDNA synthesis was performed using 200 units of Superscript™ II Reverse Transcriptase (Invitrogen Ltd., Cat. No. 18064-14), 50- 250ng of random hexamers and 0.1M DTT following the manufacturer’s guidelines.

2.2.8 Phenol:chlorophorm plasmid DNA extraction

50µl of PCR product was made up to 100μl total volume with sterile water before adding 100μl of phenol/chloroform. The samples were vortexed and centrifuged at 13000g for 5 minutes and the supernatant transferred to a new 1.5ml tube. Twice the initial volume of 100% ethanol (400µl) and 1µl of glycogen (10-20 mg/ml) was added before placing the sample at -20°C for 30minutes. The samples were centrifuged again for 10minutes 13000g before washing twice with 70% (v/v) ethanol. The pellet was air dried and re-suspended.

2.2.9 DNA sequence analysis

Sequencing reactions were performed using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Each reaction contained the appropriate primer (3.2 pmol), 4 µl of the Terminator Ready Reaction Mix, 2µl of Big Dye Sequencing Buffer (5x) and 200-500 ng double stranded DNA or 30-90ng PCR products DNA. Each reaction was made up to 10 µl with sterile distilled water. PCR cycles (25) were as follows: 96ºC for 10 s, 50ºC for 5 s and 60ºC for 4 minutes. Sequencing electrophoresis was performed using an ABI automated sequencer (Applied Biosystems). Nucleotide sequence data was edited using EditSeq package within DNASTAR (DNASTAR, Inc).