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The transformation for the in situ generation of overhangs experiments were split in half after recovery in SOC medium (half plated on ampicillin- and the other half on kanamycin-containing plates).
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The equivalent of a full FLX run was done but as two half plates on two separate dates.
Pyrosequencing generated approximately 217,000 unpaired reads (~250 nt); from a half plate on a 454 FLX instrument (Agencourt Biosciences, Beverly, MA).
A total of 5 µg of double-stranded cDNA was sequenced (half plate) on a GS-FLX Titanium sequencer at the Genome Québec Innovation Center (McGill University, Montréal, QC, Canada).
A half plate of 454 sequencing on the GS FLX Titanium platform was applied to each sample (http://www.454.com).
Another set of half seeds were plated on 1/2 MS medium containing various concentrations of NaCl (125 mM and 175 mM).
With only a small amount of sequencing (one-half plate on the GS20) in Arabidopsis, we tagged more than 15,000 genes and completely or nearly completely sequenced several hundred of the highly expressed genes.
Seeds were surface sterilized and plated on half-strength MS medium including 0.8% agar and the antibiotics needed for dual selection.
Seeds were surface sterilized and plated on half-strength MS medium including 0.8% agar and the seedlings were screened under a stereomicroscope equipped with a fluorescent lamp and green fluorescent protein (GFP) filter.
Surface-sterilized seeds were stratified for two days and plated on half-strength Murashige and Skoog medium (MS/2; 0.5x Murashige and Skoog salts with 1% sucrose, pH 5.7).
Seeds of A. thaliana (Col-0 ecotype) derived from selfed progenies of an Arabidopsis plant were surface-sterilized for 15 min in 10% bleach, washed four times with sterile water, and plated on half-strength Murashige and Skoog [ 24] medium.
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