After removal of the wire, the arteriotomy site was ligated.
The transection sites were ligated.
The Sfi I fragment containing the nfsA gene and the upstream ribosome binding site from pSV036B9 was ligated downstream of the tac promoter into the bacteriophage λ-vector λJG3J1 (Grove et al, 2003), producing λSV054, which was packaged and used to generate lysogens of the E. coli strain, UT5600 (nfsB−), as before (Grove et al, 2003).
Primer PGSU0800-vanR-PacpP-vanA PGSU0800-vanR-PacpP-vanA PGSU0800-vanR-PacpP-vanA PGSU0800-vanR-PacpP-vanA promoter regBlockith VanR binding sites was digested with NcontainingeI and ligathe into the same sites in promoter to generegionsVMCS-2.
Next, a 161-bp fragment containing both putative DAF-16 binding sites was PCR amplified with primers containing unique BamHI and NheI restriction sites and the digested product was ligated to a basal promoter in pWBI067, consisting of 398-bp upstream from the translational start, creating pWBI072.
The site of NlaIII cleavage was ligated with an Illumina-supplied adaptor using T4 DNA ligase.
Oligos harboring 13 repeats of a miR-10b-binding site or control-binding site were synthesized, phosphorylated, annealed and ligated downstream of a CMV-eGFP cassette.
Individual binding sites were cloned in pBend2 by annealing two complementary oligonucleotides and ligating them into XbaI-restricted vector.
Then, the cat-sacB fragment was ligated into PstI site of pYA4574 to form plasmid pYA4575.
The GEX Adapter 2 was ligated at the site of MmeI cleavage.
