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In the Sequential trials described above, subjects retrieved and used tools that were significantly longer than the average tool length predicted by random probing, but often still shorter than required to reach the food.
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The specificity of the assay was tested by the random probe control.
Two-colour FISH was performed by labelling 100 ng for each BAC clones with alexa fluorochromes (ChromaTide® Alexa Fluor® 488-5-dUTP 488-5-dUTP 488-5-dUTProMolecularlexa Fluor® 568-5-dUTprobeseChromaTide®s) by rAlexa priming using the Bioprim Kit (Invitrogen).
We inserted batch effects by multiplying DNA methylation values at random probes by a range of percentages from 10 100% at a range of randomly selected probes within half of the reconstitution data set, leaving the other half untouched (Fig. S4A and B ). Five iterations were performed at each level for a total of 500 comparisons.
The membrane was probed with radiolabelled DNA probe generated by random priming of a fragment corresponding to the CMV promoter, and washed in 1× SSC, 0.1% w/v SDS at increasing temperature, followed by a final wash in 0.1× SSC, 0.1% w/v SDS.
Southern blots were hybridized to [α-32P]-labeled probes generated by random priming.
The RNA was transferred to Hybond N+ membranes in 50 mM NaOH and then hybridised to dsDNA probes labelled by random priming.
The Northern blots were hybridised with DNA probes labelled by random priming in the presence of [α-P]dATP, according to the procedure recommended by the manufacturer (Random Primers DNA Labelling System; Invitrogen, Carlsbad, Calif).
The DNA was transferred to Hybond-N (Amersham) and hybridized to the [α-P]-dCTP telomeric probe labelled by random priming (Megaprime DNA Labelling System, Amersham Pharmacia Biotech, UK).
The simulated distribution of average ranks from probe sets composed from random probes was obtained by random assignment of ranks to probes from arrays.
Background subtraction was performed by subtracting the top 2% of random probe (negative control) intensity values from individual O. dioica probe intensity values.
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