For each selection round, DNA, target protein, and TGK buffer were combined in 10 μL total volume.
Amplification of the endogenous actin gene was performed as follows: either 20 ng genomic or 2 ng plasmid DNA, and 0.42 m m dNTP (GeneAmp), 0.33 µ m each primer, 1.25 U Amplitaq Gold (Life Technologies), 1.5 m m MgCl2 and 3.0 µl 10× PCR Gold buffer were combined in a final volume of 30 µl.
A total of 20 ng of DNA template, 1.25 pmols of each primer, 0.25 μ M of each deoxynucleotide triphosphate, 2.5 μ M MgCl2, 2% dimethyl sulphoxide, 1.5 U Taq polymerase (Promega, Madison, WI, USA) and the manufacturer's recommended buffers were combined in 25 μl reactions.
Phage stocks in either in LB or TM buffer were combined with the cells on ice for 15 minutes at the multiplicity of infection of approximately 10.
Cell suspensions in digestion buffer were combined with 4U of Zymolyase (Zymo Research), incubated at 37°C for 4 hours, and then stored at 4°C until ready for use.
Briefly, RIA buffer [80% phosphate-buffered saline (PBS), 20% DMEM, 2% normal rabbit serum], 100 μL cold standard (rat PRL-RP-3) or unknown sample, rPRL-s-9 antiserum (final dilution of 1 437,500 in RIA buffer), and [I]-rat-PRL (PerkinElmer, Wellesley, MA, USA; using 15,000 counts per tube diluted in RIA buffer) were combined and incubated with shaking, overnight at 4°C.
Briefly, RIA buffer (80% PBS, 20% DMEM, 2% normal rabbit serum), 100 μL cold standard (rat PRL-RP-3) or unknown sample, rPRL-s-9 antiserum (final dilution of 1 437,500 in RIA buffer), and [I]-rat-PRL (PerkinElmer, Wellesley, MA; 15,000 counts diluted in RIA buffer) were combined and incubated overnight at 4°C.
Protein extracts containing 35 μg of total protein, recombinant HvAOS activity (4 nkat; [ 40], and sodium phosphate buffer (50 mM; pH 7.0) were combined in a final volume of 200 μl.
Probes (4 ng/µL) were combined in hybridization buffer supplied with the dyes, denatured for 10 min at 72°C in 50% formamide in 2X SSC, and kept on ice for 30 min before application to the slides.
200 pmoles of purified Cy3- and Cy5-labelled aRNAs were combined in a buffer containing 2× SSC, 0.08% SDS and Liquid Blocking Reagent (GE Healtcare), and were dispensed over the microarray surface, and incubated at 55°C overnight with agitation.
To create fork and full duplex substrates, corresponding oligo-nucleotides were combined in annealing buffer (50 mM Tris-HCl pH 7.0 and 25 mM KCl) in a 1 2 ratio of labeled to unlabeled oligonucleotide, heated to 90 °C for 10 minutes and cooled slowly to room temperature.
