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Example sentences for: stratagene
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Purified TOP1 was pre-incubated with GST or GST-BTBD2 for 20 min before addition of the DNA substrate, which was either an end-labeled 36 bp fragment containing a single strong TOP1 cleavage site [ 24 ] , or a 161 bp PvuII-HindIII fragment of pBluescript (Stratagene) [ 25 ] ). End labeling of the DNA substrates was as previously described [ 24 25 ] . Cleavage reactions were continued for 20 min before stopping with 0.5% SDS.
Prehybridization and hybridization were performed at 42°C and using Quick Hyb ®(Stratagene) as hybridization solution.
To construct the expression plasmid encoding C-terminal-myc-tagged, full length BTBD2 (m-BTBD2F), the entire coding region of BTBD2 (1-1575 bp) was amplified with primers (Upper primer: 5' ATGGCGGCGGGTGGGAGCGGCGGGCGTGCGTCGTG-3', Lower primer: 5'-GGTGTAGAAGATGACCTCGGGGATCTGGCCGTCCTCCAC-3') containing BamH I and Hind III cleavage sites and inserted into pCMV-Tag 5 vector (Stratagene) cut with BamH I and Hind III.
The cDNA library (named Bird_Rao_ Meloidogyne_incognita _J2) was constructed using the Zap Express cDNA Synthesis Kit and Gigapack III Gold Cloning Kit, 200403 (Stratagene, Cedar Creek, TX).
Oligonucleotide primers for the PCR reaction were based on the published sequence of murine Rab24 [ 24 ] . Kpn1 and BamH1 restriction sites were added to the 5' and 3' primers respectively, to facilitate cloning of the PCR product in frame with a 5' sequence cassette encoding a myc epitope (EQKLISEEDL), previously introduced into the pCMV5 expression vector [ 78 ] . The Rab24 cDNA was altered by overlap-extension PCR [ 79 ] , using appropriate mutator oligonucleotides and Pfu DNA polymerase (Stratagene Inc., La Jolla, CA), so as to introduce amino acid substitutions at D123 (D123I), T120 (T120A), S22 (S22N), S67 (S67Q), and a deletion of residues 127 through 130 (ΔEDRR).
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