Editing Template swiching cDNA library construction

== Primer sequences ==

Primers for first strand synthesis: 

TS oligo (SfiI site bold) 5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCrGrGrG-3' (rG = ribonucleotide G); 

iso3TS oligo (SfiI site bold) 5'-iCiGiCAAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCrGrGrG-3' (iC = iso-dC, iG = iso-dG); 

Oligo-dT primer (SfiI site bold) 5'-TAGAGGCCGAGGCGGCCGACATGTTTTGTTTTTTTTTCTTTTTTTTTTVN-3'. 

Primers for LD-PCR: iso3TS oligo as 5'PCR primer; 3'PCR primer: 5'-TAGAGGCCGAGGCGGCCGAC-3'.



== First strand cDNA synthesis ==



First strand cDNA synthesis was performed in 106 μl reactions assembled in the following manner. For each sample, the following components were placed in a 200 μl PCR tube on ice: 52.5 μl RNA sample, 8.4 μl oligo dT primer (10 μM), 8.4 μl TS oligo or iso3TS oligo (10 μM), 69.3 μl total volume. RNA and primer mixes were denatured at 65°C for 2 min and then removed to ice. The following reagents were then added to each reaction tube: 20 μl 5× first strand buffer (Powerscript [Clontech] RT buffer: 250 mM Tris (pH 8.3), 30 mM MgCl2, 375 mM KCl, 20 mM DTT), 2 μl DTT (100 mM), 10 μl 50× dNTP (10 mM), 5 μl SuperScript II (Invitrogen, Carlsbad, CA). First strand reactions were incubated at 42°C for 2 hr.



== First strand cDNA cleanup ==

==  ==

A cleanup step was performed for first strand cDNA samples using Machery-Nagel NucleoSpin Extract II kits following the manufacturer's protocol and the total recovered elution volume was 80-85 μl per sample.



== cDNA amplification by Long-Distance PCR (LD PCR) ==



For PCR amplification of full length cDNAs, 80 μl of cleaned-up first strand cDNA was used as template in 100 μl PCR reactions including the following reagents: 1× Advantage 2 PCR Buffer (Clontech), 0.2 mM dNTPs, 0.12 μM 5'PCR primer (iso3TS oligo), 0.12 μM 3'PCR primer, and 1× Advantage 2 Polymerase Mix. PCR amplifications were performed using an MJ Research Dyad (MJ Research, Waltham, MA, USA) with the following program parameters: an initial hold at 95°C for 1 min, followed by 27 cycles of 95°C for 5 s, 65°C for 5 s, and 68°C for 6 min. After completion of a PCR reaction, a 5 μl aliquot of each sample was removed and run on a 1.1% agarose/TAE gel containing 0.5 μg/ml ethidium bromide alongside a DNA MW marker such as the Fermentas Mass Ruler (Fermentas, Glen Burnie, MA, USA).



== LD PCR reaction clean up ==



LD PCR amplified cDNA samples were cleaned up using NucleoSpin II kits following the manufacturer's protocol and the total combined eluate was approximately 32 μl.



== SfiI digestion of cDNA ==



SfiI digestion was performed in 40.5 μl total volumes containing 32 μl cDNA, 4.05 μl 10× SfiI buffer 4.05 μl SfiI enzyme, and 0.4 μl 100× BSA. Digestions were incubated at 50°C for 2 hrs before proceeding to cDNA size fractionation. SfiI buffer and enzyme were obtained from NEB (Ipswich, MA, USA).



== 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]]