In this case, the localization protocol should be reversed: anchor nodes obtain RSS values for packets sent by the UN, and then send those measurements back to the node (or to a central processor) to estimate the UN position.
The features of this signal resemble those of a reverse signal anchor sequence (Goder and Spiess, 2001).
To confirm directed insertion of the PUbECFPnls-SV40 cassette into the AaegTRPA1 locus, a diagnostic PCR product (no wild-type band, 2075 bp mutant band) was amplified using a forward primer anchored outside the boundary of the left homologous arm (5'-CATGGGACAATTTGGCGTAGGCAGTAT-3') and an ECFP reverse primer anchored inside the inserted cassette (5'- AGATCTCGACCCAAGAAAAAGCGGAAG-3').
Forward and reverse primers anchored at the predicted start and stop codons were designed (Table 1).
Richie describes this as "de-legacy" activity, suggesting that it is more important to "stop or reverse un-innovation anchored on the wrong side of history" than to only look forward.
2 µg of total RNA was used for reverse transcription using Anchored Oligo- dT 23 (Sigma) as primer fOligo- dT 23d Sigmaesis using the RT-asV kit (Roche).
First strand cDNA was synthesized in reactions containing 2 µg of total RNA pooled from 5 H. virescens midguts using PowerScript™ reverse transcriptase (Clontech), anchored oligo dT 20 primer (Invitrogen) and other reaction components in a 10 µl reaction.
cDNA was reverse transcribed using anchored oligo dT primer and Superscript II (Life Technologies) following the manufacturer's instructions.
First strand cDNA was generated using reverse transcriptase, an anchored dT primer, and 700 ng total RNA from each of the six samples.
Molecular analysis of all expressed TRB gene rearrangements was subsequently conducted using an unbiased template-switch anchored reverse transcription PCR as described previously [ 37– 37].
[αP] dCTP-labelled single-strand cDNAs were synthesized from 20 μg of total RNA by reverse transcription using anchored oligo dT 20VN primer and Superscript III Reverse Transcriptase (Invitrogen) following standard protocols.
