Natarajan et al. BMC Genomics 2010, 11:606 http://www.biomedcentral.com/1471-2164/11/606 Page 10 of 14 Figure 6 Semi-quantitative RT-PCR analysis of expression of selected genes. Expression of seventeen genes involved in oil biosynthesis were analysed by Semi-quantitative RT-PCR in flower (F), mature leaves (L), root (R), and developing seeds (S) of J. curcas. Actin was used as an internal control. Name of the genes studied, the primers used for PCR amplification, and the size of the PCR amplified fragments are given in Table 3. adaptor. The sense strand of the double strand tester cDNA which is phosphorylated at the 5’end was destroyed by treating it with lambda exonuclease. As a result only the anti-sense strands are retained. Double strand driver cDNA was prepared by low cycling PCR using phosphory- lated driver3 primer and driver5 primer (TCCTAA GGTAGCGAGGCCATTACGGCCGGG) which is com- plementary to the 5’driver adaptor. The anti-sense strand of the double strand driver cDNA which is phosphorylated at the 5’end was destroyed by treating it with lambda exo- nuclease. As a result only the sense strands are retained. Anti-sense strands from tester cDNA and sense strands from driver cDNA were mixed and hybridized in 1× hybridization buffer (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 0.2 mM EDTA) at 68°C for 6 h. The double strand hybrids formed during hybridization were removed by hydroxyapatite chromatography [59] for normalization of the single strand cDNA population. The normalized single strand cDNAs were converted to double strand cDNA and amplified by FailsafeTM PCR system (Epicentre Biotechnologies, USA) using amplifi- cation primer (CAGTGGTATCAACGCAGAGT) for which the binding sites are present both in the 5’ and 3’ end of the cDNA and it is specific to the tester cDNA only (underlined in tester3 and tester5 primers used for cDNA synthesis). The amplified cDNA was digested with SfiI restriction enzyme and cloned in modified pBluescript II SK- in which SfiA (GGCCATTACGGCC) and SfiB (GGCCGAGGCGGCC) sites were introduced between EcoRI and XhoI. The ligated plasmids were transferred to E. coli DH10B-T1R (Invitrogen, USA) to construct the cDNA library. Normalization Efficiency Normalization efficiency was strictly monitored by per- forming parallel normalization experiment. Chloramphe- nicol resistance gene with same adaptor and primer sequences was used as reporter gene. In the parallel nor- malization, the reporter gene was added to the cDNA population as internal control at 1.0% redundant rate. Cloning of cDNA using modified pBluescript II SK- was performed. Normalization efficiency was determined by plating the cDNA library on LB plates containing chloramphenicol. Quality Control of cDNA Library Ninety six colonies from the cDNA library were ran- domly selected for quality control experiment. Cells from these 96 colonies were used for colony PCR as well as plasmid DNA isolation for sequencing. Colony PCR was performed using M13 forward (GTAAAACGACGGC- CAGT) and M13 reverse primer (CAGGAAACAGC- TATGAC). The PCR products were run on 1.0% agarose gel with 1.0 kb ladder as marker (Genie, Bangalore, India). Clones without cDNAs (empty clones) will give PCR product size of 226 bp and clones with cDNA will give variable PCR product size higher than 226 bp depending on the length of the insert. Based on this, approximate size of cDNA in each clone was determined. For plasmid DNA isolation, the cells were inoculated in 5 ml LB Broth supplemented with 50 mg/L ampicillin and incubated at 37°C at 200 rpm for 16 hours. Plasmid DNA was isolated from 1.5 ml of culture using Plasmid Mini- prep Kit following manufacturer’s protocol (Biobasic Inc, Canada). About 200 ng of plasmid DNA was used for sequencing using M13 reverse primer and BigDyeTM Terminator v3.1 Cycle Sequencing Kit in 3130xl Genetic Analyzer (Applied Biosystems, CA, USA). These sequences were annotated by using BLASTX algorithm and non-redundant database at NCBI [61]. Sequencing of Expressed Sequence Tags (ESTs) The cDNA library was serially diluted and plated on Luria Bertani (LB) agar plates supplemented with 50 μg/ml ampi- cillin and incubated for 16 hours at 37°C. Well separated single colonies were randomly selected and patched on LB agar plates supplemented with 50 μg/ml ampicillin, and incubated overnight at 37°C. The cells from patched colo- nies were inoculated in 5 ml LB Broth supplemented with 50 mg/L ampicillin and incubated at 37°C at 200 rpm for 16 hours. Plasmid DNA was isolated from 1.5 ml of culture using Plasmid Miniprep Kit following manufacturer’s