Hela genomic dna

Methylation-specific polymerase chain reaction (MSP) was conducted using two primer pairs; each specifically designed to attach to methylated or unmethylated strands. Segments were amplified in a total volume of 12.5 µL (Table 1) according to reactions profiles detailed in Table 2. Sequence, relative length, GC content and annealing temperature of primers are shown in Table 3 [27 (link)].
CpG-methylated HeLa Genomic DNA (New England BioLabs; Cat.#N4007S) was utilized as positive control for methylation, negative control was fully unmethylated DNA (EpiTect PCR control DNA; Qiagen). Two separate temperature gradients were performed to assess optimal annealing temperature for both forward and reverse primers (miR-137; 57 °C, miR-342; 63 °C). The specific band size observed was 140 bp and 210 bp, respectively. PCR products were run on 2% agarose gels and visualized via EtBr fluorescence (Figure 1).

Oligonucleotides were purchased from Biomers or Metabion, HeLa genomic DNA and Taq 2x master mix was bought from New England Biolabs, dNTPs were either from Roche or Fermentas, Phusion DNA polymerase was purchased from Thermo Scientific, Platinum Taq and AmpliTaq Gold DNA polymerases from Life Technologies, DNase I, SphI, and HindIII from Fermentas, the Gel Extraction and EpiTect MSP kit from Qiagen and used according to their manuals. KlenTaq and its respective mutants were recombinantly expressed in E. coli BL21 (DE3) and purified with Ni-IDA as previously described[63] (link). Enzyme purity and quantity were determined by SDS-PAGE using an albumin standard dilution curve. KlenTaq variants were stored in 50 mM Tris-HCl (pH 9.2), 16 mM (NH₄)₂SO₄, 0.1% Tween20, 2.5 mM MgCl_2, 50% glycerol at −20°C. For real-time PCR a Chromo4 instrument from Bio-Rad or a Roche LightCycler 96 or 480 system was used. SYBR green I was purchased from Fluka. PCR was performed in the GeneAmp PCR System 9700 from Applied Biosystems. Genomic DNA samples were bought from NIBSC (Prothrombin Mutation G20210A, Human gDNA, 1st International Genetic Reference Panel 2005 - WHO International Standard or Reference Reagent Product Number 05/130).