Fig 1: Rolling circle amplification products on denaturing 15%TBE urea gel. The templates were annealed with a single spanning primer. The ligation process with T4 DNA ligase (NEB, Cat# M0202S) was mediated by the phosphorylated upstream template. Last, ?29 DNA polymerase was used to polymerize the oligos (NEB, Cat# M0269L). The rolling circle amplification (RCA) products were hybridized with spacer complementary to produce tandem repeats of ssDNA alternating with dsDNA to increase the chance of interaction with target Salmonella. Few controls have been added to ensure the quality of the RCA products.
Fig 2: T4 RNA Ligase 2 catalyzes RNA-templated DNA-to-DNA ligation.(A) Left panel: ligase screen for RNA-templated DNA–DNA ligation activity. Ligases were incubated with an unlabeled single-stranded DNA (left) or RNA (right) template hybridized to a common pool of 5' end 32P-labeled (circled P) DNA oligonucleotides for 1 hr. Both T4 DNA ligase and T4 RNA ligase 2 (Rnl2) catalyze RNA-templated DNA–DNA ligation. Also note the inability of Rnl2 to ligate >2 oligos on the DNA template. For both templates, ligases are left to right: Tth DNA ligase (Thermo), Tsc DNA ligase (Prokaria), Thermostable DNA ligase (Bioline), T4 DNA ligase (NEB), T4 Rnl2 (NEB), E. coli DNA ligase (NEB). The three rightmost lanes are 32P-oligos only, 32P-labeled RNA template, and a 32P-labeled low-molecular weight DNA ladder (NEB, N3233S). Right panel: Rnl2 and T4 DNA ligase time course for oligos hybridized to the RNA template. Templated ligation products (–x2 through –x5); non-templated ligation product (*–x6). (B) Rnl2 can join multiple 32P-labeled ligamers each looping out sections of the template but only when they are adjacently hybridized. Gray or white square: ligamer present or absent, respectively. No template (-T); no enzyme (-E). (C) Cis- and trans-transcript hybridization and ligation using a ligamer (W) spanning 1046 nt common to two RNAs (XWY and VWZ). Template concentrations (nM) were as indicated above each lane (ranging from 0.01 to 100 nM), ligamers were held constant at 10 nM. Left panel, phosphoimage; right panel, SybrGold stained. (D) The ability of SeqZip to accurately report on relative input RNA concentrations was investigated using various ratios of two RNAs (XWZ and VWY) and a six ligamer pool. Observed product ratios were calculated from radioactive PCR band intensities.DOI: http://dx.doi.org/10.7554/eLife.03700.006
Supplier Page from New England Biolabs for T4 DNA Ligase
Unit Definition:One NEB unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of Lambda DNA in 30 minutes at 16°C in 20 µl of the above assay mixture and a 5´ DNA terminiconcentration of 0.12 µM (300 µg/ml).