Editing Template swiching cDNA library construction (section)

== cDNA size fractionation by gel electrophoresis ==



cDNA was fractionated by molecular weight using preparative DNA gel electrophoresis after SfiI digestion. Digested cDNA samples were loaded (using a loading buffer containing bromophenol blue) on a 2% low melt agarose (NuSieve GTG, Cambrex, Rockland, ME, USA) gel containing 0.5 μg/ml ethidium bromide. Electrophoresis was performed at a low voltage setting (5-8 V/cm) until the bromophenol blue dye had migrated approximately 4 cm. Gels were photographed and the lanes excised from the 400 bp molecular weight marker up to the sample well. DNA was isolated from the gel slice using a Qiagen MinElute kit (Valencia, CA, USA) and 6 sample volumes of buffer QG. Elution was performed using 10 μl of elution buffer. Ligations into pDNR-LIB (Clontech) were performed immediately following cDNA size fractionation and elution according to the method specified in the SMART cDNA library construction manual. Sequencing of gland-derived samples was accomplished using traditional Sanger sequencing of individual clones. The digested, fractionated cDNA samples from rhizome tips and elongation zones were purified using QIAquick PCR purification kit (Qiagen). 5~10 μg purified cDNA from each rhizome sample was used in construction of a 454 Genome Sequencer FLX [21-23] fragment library using Titanium series reagents [http://www.454.com http://www.454.com] followed by sequencing on a GS FLX instrument.
[[Category:Antibodies]]