Once the specificity of the primers for the nematode community under study was determined

e month old D4A2 plants grown in tissue culture jars. Leaves were sprayed with 12 mL of 0.7 M ethanol and the jar lids replaced. For Arabidopsis one month old plants selected on petri dishes and then PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189597 grown in soil were sprayed with 12 mL of 0.7 M ethanol and covered with a plastic bag to maintain the presence of ethanol vapour. The plants were then left for 4 days to allow time for I-SceI expression, the generation of DSBs and their subsequent repair. After 4 days leaf tissue was sampled. Plasmid construction and plant transformation pAlcR:ISceI. The AlcR expression cassette containing the 35S promoter, AlcR ORF, and nos terminator was isolated as a NcoI/ HindIII fragment from pbinSRN. This cassette was blunt ended using the Klenow fragment of DNA pol I and cloned into SmaI cut pGreen0179 to generate pG.AlcR. The I-SceI RGFA-8 chemical information coding region was excised from pCISceI and inserted between the alcA:35S promoter and nos terminator in Alc-pUC, using BamHI. Primers AlcF_NcoI and AlcR_NcoI were then used to amplify the I-SceI expression cassette and the product was ligated into pG.AlcR using NcoI to generate pAlcR:ISceI. pdao1. The 35S terminator from pPRVIIIA::neoSTLS2 and 35S promoter from p35S were cloned into pGreen0029 using HindIII/BamHI and NotI/XbaI respectively. The dao1 coding sequence was amplified from pVC_RLM_1qcz using primers dao1F and dao1R_XbaI, the PCR product was then digested with XbaI and cloned into the pGreen0029 vector containing the 35S promoter and terminator, thus generating pG.dao1. A multiple cloning site containing two I-SceI restriction sites flanking HindIII and NotI sites was generated by annealing two complementary oligonucleotides I-SceIMCS1 and I-SceIMCS2. This double stranded MCS had 4 bp overhangs at each end allowing ligation into SacI and XhoI cut pGreen0029, generating pG.MCS. The dao1 expression cassette was excised from pG.dao1 with HindIII and NotI and cloned into HindIII/NotI digested pG.MCS to generate pdao1. Transformation was performed using the pGreen system of binary transformation vectors. Transgenic tobacco lines were generated using a standard leaf disc method. Transgenic Arabidopsis lines were generated using the simplified floral-dip method with a rapid selection protocol. Putative D line and A line transformants were confirmed by PCR using primer pairs dao1F2/dao1R2 and AlcRF1/AlcRR1 respectively. PCR TAIL-PCR was undertaken as described using degenerate primer AD2 and pdao1 T-DNA specific primers dao1T1, dao1T2 and dao1T3. Standard PCR was performed using taq polymerase, using suggested PCR conditions. DSB PCR products were amplified with LongAmp taq DNA polymerase using suggested PCR conditions, primers DSBF1 and DSBR1, an annealing temperature of 59uC and 40 ng template DNA. Non-induced, induced/undigested and induced/digested DNA was used as template. For digested template, 2 mg genomic DNA was digested overnight at 37uC using 20 u HincII in a 20 mL reaction and purified using a PCR purification kit according to manufacturer’s instructions. Single molecule PCR was performed using LongAmp taq DNA polymerase and HincII digested DNA as template. Reactions were 2 mL in volume and contained 0.3 mM dNTPs, 0.4 mM primers, 0.2 u LongAmp taq DNA polymerase, 16 LongAmp buffer and 110130 pg template DNA for tobacco or 1 pg template DNA for Arabidopsis. Reactions were overlayed with mineral oil to prevent evaporation. Cycle conditions were as follows: Initial denaturation 95uC 309 then 45 cycles of 95uC