PCR Methylation specific PCR - Bacterial DNA

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. However, non-specific product amplification and primer-dimer formation during set-up are major causes of PCR failure. Nevertheless, high-quality DNA polymerase and optimize reaction buffers will certainly lead to a successful PCR reaction.

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2 years ago

2 years ago by Daniel McKenzie United Kingdom

How “strong” is the PCR product of methylation specific PCR?

Does anyone know how “strong” the PCR product of methylation specific PCR is? I kept my PCR products at 4C for about 3 weeks and then at room temperature for another week. Will I be able to use them for sequencing?

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Found 6 matching solutions for this experiment

Sodium bisulfite

Sigma-Aldrich

Protocol tips
The methylation status of the target genes was determined using a methylation-specific PCR with primers specific for the methylated and unmethylated alleles of each gene after treating the genomic DNA with sodium bisulfite –. The primer sequences, annealing temperatures, and the expected sizes of PCR products are summarized in . Briefly, 1 µg of DNA was denatured with sodium hydroxide and modified with sodium bisulfite and DNA samples were purified using Wizard DNA purification resin (Promega). The sample DNA was treated with sodium hydroxide again, precipitated with ethanol, and resuspended in distilled water. All PCR amplification steps were carried out using reagents supplied in a GeneAmp DNA Amplification Kit with AmpliTaq Gold (PE Applied Biosystems) on PTC-100 (MJ Research).
Protocol tips
The DNA was sheared to ~2 kb using a Bioruptor (Diagenode). Bisulfite conversion was performed using the EpiTect Bisulfite kit (Qiagen), following the manufacturer's guidelines on 'Sodium Bisulfite Conversion of Unmethylated Cytosines in Small Amounts of Fragmented DNA'.
Protocol tips
S-adenosylmethionine (SAM) is unstable at (pH 7.5), 37°C and should be replenished in reactions incubated longer than 4 hours.
HaeIII Methyltransferase

New England BioLabs

Protocol tips
S-adenosylmethionine (SAM) is unstable at (pH 7.5), 37°C and should be replenished in reactions incubated longer than 4 hours.
dam Methyltransferase

New England BioLabs

Protocol tips
S-adenosylmethionine (SAM) is unstable at (pH 7.5), 37°C and should be replenished in reactions incubated longer than 4 hours.
Protocol tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.
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