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Example sentences for: linkers
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RNA was reverse transcribed into cDNA using random hexamer primers in the presence of 5-aminoallyl-dUTP; Cy3 and Cy5 dyes were covalently coupled to the incorporated aminoallyl linkers in a subsequent labeling reaction.
We reasoned that a Tn 5 transposon [ 11 12 ] could be designed that would generate GFP fusion proteins with relatively short linkers (~7 amino acids) between the GFP and the host protein analogous to GFP fusion proteins that have already been shown to function [ 6 8 ] . To test this approach, we targeted the G protein subunit α s and the glutamate receptor subunit GluR1.
Sodium salts of each compound were titrated into assays of purified Fhit [ 16 ] with 1.8 μM fluorescent substrate, Appp-S-(4-4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacine-3-yl)methylaminoacetyl (ApppBODIPY) [ 10 ] , and competitive K i values were obtained by calculating the inhibitor concentration-dependence in reduction of k cat / K m (apparent) for substrate hydrolysis [ 10 ] . As shown in Table 1, inhibitors 2b and 3b with two-carbon diol linkers were either only as inhibitory as the natural substrate 1 or substantially less so.
The first protocol uses a ligation-mediated PCR strategy (LM-PCR) in which ChIP DNA is ligated to short DNA linkers, and then PCR amplified using the linker sequence for priming [ 4 ] . The second uses a random amplification strategy (R-PCR) in which template DNA is first copied using a specialized primer with degenerate 3' sequence and known 5' sequence, and then PCR amplified using the known 5' sequence for priming [ 5 8 9 ] . In both cases, the PCR steps yield quantities of nucleic acid sufficient for microarray analysis.
This is analogous to the use of the bovine pancreatic trypsin inhibitor for internal insertions [ 21 ] . The transposons described here could potentially be improved upon by optimizing the length and flexibility of the linkers between the target and the GFP.
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