For the isolation of DNA from elongated plants a modified BET bromodomain inhibitor buffer with higher salt concentration was used. The amount and quality of DNA was estimated on a Nanodrop spectrophotometer. Southern blot analysis A total amount of 6 ug gDNA was digested overnight at 37 C with 140 U EcoRV and XbaI in independent reactions, each enzyme providing only one restriction site within the T DNA of the binary vector. The digested DNA was separated on a 1% Inhibitors,Modulators,Libraries agarose gel for 17 h at 23 Volt. DNA was blotted overnight onto a Gene Screen Plus Hybridization Transfer Membrane using the capillary transfer method. A gene specific probe for the hptII gene was amplified with the primer pair and radiolabeled with dCTP using the Rediprime II DNA Labeling System according to the manufacturer s instructions.
RNA isolation and qRT PCR Tissue was harvested from rosette stage leaves and ground Inhibitors,Modulators,Libraries in liquid nitrogen to a fine powder. RNA isolation was performed with a salt precipitation Inhibitors,Modulators,Libraries method modified from the US patent of Gentra Systems, Inc. publication No. 5973137 and adapted for N. attenuata tissue. Ap proximately 150 300 mg ground and frozen tissue was dissolved in 900 uL cell lysis buffer and shortly mixed. Per sample 300 uL protein precipitation buffer was added and the tubes inverted ten times. Samples were incu The supernatant was collected and extracted with 500 uL chloroform isoamylalcohol mix. After centrifuga tion the upper aqueous phase was col lected and nucleic acids precipitated with 1 volume isopropanol for 15 min at room temperature.
Nucleic acids were pelleted in a table top centrifuge, washed twice with 400 uL 70% ethanol and air dried for 5 min. The final pellet was dissolved in 50 uL nuclease free water. The nucleic acid was DNAse treated using the TURBO DNA free kit according to the manufac turers instructions. Quality and amount of the remaining Inhibitors,Modulators,Libraries RNA was determined using a 1% agarose gel and a Nanodrop spectrophotometer. The ab sence of genomic DNA was tested with 20 ng RNA in a 35 cycle PCR programm with the same primers as for qRT PCR. 4 ug of total RNA was reverse transcribed with oligo 18 primers and the SuperScript II reverse transcriptase enzyme. Quantitative Real Time Inhibitors,Modulators,Libraries PCR was performed with 1 10 diluted cDNA on a Mx3005P QPCR System with either a SYBR Green based PCR Master Mix or a qPCR Core kit for SYBR Green.
For amplification the following primers were used ICE 94F The used pro gram was 95 C for 10 min followed selleck screening library by 40 cycles of 95 C for 15 s, 60 C for 1 min and 1 cycle of 95 C for 15 s, 60 C for 30 s, 95 C for 15 s as dissociation curve. For relative gene expression analysis the comparative cycle threshold method was used. Gene expres sion was shown as log2 relative to N. attenuata actin as the reference gene Bisulfite genomic sequencing DNA methylation analysis was performed by the bisul fite sequencing method.