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Newcastle's winning sequence was ended at the fifth time of asking at Swansea on Wednesday evening, and while that came as a blow, the manager is hopeful of a response.
He will become Leeds' fourth permanent boss in just five months after Milanic's 32-day, six-game winless sequence was ended following a 2-1 home defeat by Wolves on Saturday.
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After 48 h of culture, transient gene expression was determined by western blotting, because the inserted sequences were ended with a terminal codon before the EGFP gene.
PCR primer (200 ng) for probe sequence was end-labeled with Pγ-ATP and T4 polynucleotide kinase (New England Biolabs, Beverly, MA, USA), and was column purified.
Four oligonucleotides, RATZEB1, RATZEB2, RATZEB3 (5'-GCTGTATTTAAG andATCCG-3') and RATZEB4 (5'-ATACAGCTGAAACCATGC-3') corresponding to PRAT sequence were end-labelled with [γ-32P]dATP in phosphorylation reaction with T4 polynucleotide kinase (Fermentas).
Complementary oligonucleotide probes specific to small RNA sequences were end labeled with γ32P-ATP using OptiKinase (USB Corporation) [46].
DNA oligonucleotides complementary to small RNA sequences were end-labeled with γ-32P-ATP using T4 polynucleotide kinase (NEB), and these oligonucleotides were used as the probes (Table S17).
DNA oligonucleotides complementary to miRNA sequences were end-labeled with γ-32P-ATP using T4 polynucleotide kinase (TaKaRa, Dalian, China).
DNA oligonucleotides complementary to miRNA sequences were end labeled with γ-P-ATP using T4 polynucleotide kinase (TaKaRa, Dalian, China).
DNA oligonucleotides complementary to miRNA sequences were end-labeled with r-P-ATP using T4 polynucleotide kinase (NEB, Beijing).
DNA oligonucleotides complementary to miRNA sequences were end labeled with γ-P-ATP using T4 polynucleotide kinase (New England Biolabs).
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