Molecular imager pharosfx plus

The in gel hybridization was performed as earlier described [56] (link). We used HindIII to generate long TRFs in order to minimize effect of telomere length and heterogeneity on signal detection. For the control reactions, the DNA was pre-treated with 30 units T4 DNA polymerase (New England Biolabs) for 30 min at 37°C to remove the 3′ G-overhangs. The hybridization signals were scanned using the Molecular Imager PharosFX Plus (BioRad) and quantified with the Image Lab software (BioRad). To correct for loading differences, the hybridization signals were normalized to the ethidium bromid staining of the agarose gels. The G-overhang signal was obtained after subtracting the signal from the T4 DNA Pol pretreated samples. Finally, the G-overhang signal of the wild type samples was set to one and all other samples were normalized to this value. We usually perform quantitative comparisons of samples run in the same gels to minimize experimental variation.

The V3 regions of bacterial 16S rRNA genes were amplified using primer pair 341F-GC and 518R (75 (link)). The 50-μl PCR mix contained 1 × ThermoPol buffer, 0.2 μM each primer, 200 μM dNTPs, 30 μg BSA, 1.25 U Taq DNA polymerase (New England Biolabs, Ipswich, MA, USA), and 2 μl of template. The PCR was performed in two rounds by adding 1 μl of amplification product from the first PCR (PCR1) into the second PCR (PCR2). The PCR amplifications were performed as follows: 95°C for 3 min, 35 (PCR1) or 15 (PCR2) cycles of 95°C for 15 s, 55°C for 30 s, and 68°C for 30 s, and a final extension of 68°C for 7 min. Equal amounts of PCR amplicons were separated on a 10% (wt/vol) polyacrylamide gel with a denaturant gradient ranging from 30% to 70% to a maximum of 20 μl for samples with low PCR yield. The gels were run as previously described (76 ) for 15 h at 85 V in a DGGEK-2401 denaturing gradient gel electrophoresis (DGGE) system (C.B.S. Scientific Company, San Diego, CA, USA), stained with SYBR green I DNA stain (Invitrogen) for 1 h, and scanned using the Molecular Imager Pharos FX Plus (Bio-Rad).

Glycosylase assays were done in the quadruplex reaction buffer, which contains 20 mM HEPES-KOH pH 7.4, 100 mM KCl, 1 mM EDTA, 0.1 mg/ml BSA and 1 mM DTT. In the case of Na⁺-coordinated quadruplexes, HEPES-NaOH and NaCl were used in the buffer. Substrate concentration was typically 10 nM unless otherwise specified. Enzymes and substrates were incubated at room temperature or 37°C as indicated. To measure glycosylase plus lyase activities, reactions were quenched by adding an equal volume of FE buffer (96% formamide, 20 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol) directly. To measure only the glycosylase activity, reactions were terminated by adding NaOH to a final concentration of 0.33 N and heated at 95°C for 4 min. An equal volume of FE buffer was added to the reactions before loading on to a 12% urea gel for separation. The gel was dried and exposed on a phosphorimager screen. Finally, bands from the screen were scanned by Molecular Imager PharosFX Plus (Bio-Rad Laboratories) and quantified by Quantity One software (Bio-Rad Laboratories).