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Primers were designed (Eurofins Genomics) such that it was possible to generate a 742 bp long sequence of linear dsDNA from the pUC19 vector encompassing the lacZα gene. The short nucleotide sequence was generated through PCR (35 cycles) with 1 ng pUC19 plasmid using 2x MyTaq Red Mix (Bioline) at an annealing temperature of 66°C and the band A new cloning vector pN15L is described. It is a linear 13.8 kb plasmid based on the coliphage N15 mini-replicon. The vector capacity exceeds 50 kb and the copy number is 250 per Escherichia coli The PCR product was then digested with EcoRI and SacI and ligated overnight at 16°C with T4 DNA ligase with the similarly digested pUC19 vector backbone, resulting in the creation of pUCup1. The gfp gene was amplified from the plasmid PROBE-NT' [ 10 ], and was ligated into pUCup1 using the SacI and KpnI restriction enzymes to construct pCGFP1. The pBluescript vectors, e.g., pBluescript II KS/SK (+ / -) (3 kb), are phagemids derived from pUC19 and have essentially similar configurations as pGEM-Zf except that they have T7 and T3 promoters, respectively, flanking the MCS which is composed of 21 unique restriction sites ( 95 ). The double-stranded products were digested with XhoI and cloned in the SalI site of a pUC19-derived vector conferring spectinomycin resistance and lacking BpiI and BsaI sites. For construction of the preassembly vectors pL1-TA1 to 3, a lacZ α fragment was amplified using primers ecvprac1/11, ecvprac18/19 and ecvprac23/24 (sequences given in This study explored the impact of polystyrene (PS) particles with sizes of 75, 90, 100, 1000, and 10000 nm on the HGT (via transformation) of ARGs mediated by pUC19, pSTV29, and pBR322 plasmids into Escherichia coli cells. PS particles with sizes ≤100 nm impacted the transformation of ARGs, but large particles (1000 and 10000 nm) showed no The resulting DNA fragments were digested with Sau3A1, passed over a ChromaSpin-200 size fractionation column (BD Biosciences), and ligated into the BamH1 site of puc19 vector (New England Biolabs). Standard chromatin immunoprecipitation (ChIP) assays were done as previously described for tissues ( 24 ). Open in new tab Download slide. smGFP (pUC19-smGFP) was used as a control. White bars = 50 μm. (C) Subcellular localization of GmRIC1 and GmRIC2 in Nicotiana benthamiana leaf epidermal cells. Each fusion protein was expressed by agroinfiltration of N. benthamiana leaves. Empty vector (pBIN35S-GFP)-transformed cells are shown as a control. The pUC19 plasmid reference sequence is downloaded from NCBI Nucleotide. After the re-squiggle, we normalize the raw signals of each read separately by using median shift and median absolute deviation (MAD) scale ( Stoiber et al., 2016) as follows: signalnorm = signalraw - median(signals) MAD ( signals), (1) Click the circle icon to see the annotation of your GFP sequence with the primers. Print this out and put in your notebook. From this annotation cutting the GFP restriction sites and 'pasting' into pUC19, a plasmid vector. Plasmids are extra-chromosomal circular pieces of DNA found naturally in bacteria such as E. coli. We (mankind It is reported here that the integration of IS10-related DNA sequences into the pUC19 cloning vector and its derivative occurred with considerable frequency in E. coli strains JM107 and DH10B, with duplication of a 9-bp segment (TCTAAAGTA). Residual insertion sequence elements (IS elements) in Escherichia coli strains that are commonly used for DNA cloning are known to cause cloning a