SnaBI NEB#R0130

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	For Ifng methylation analysis, 84 ng of DNA was incubated in enzyme buffer with or without 5 U of SnaBI (NEB), digested for 1 hour, and heat inactivated.

Protocol tips
For Ifng methylation analysis, 84 ng of DNA was incubated in enzyme buffer with or without 5 U of SnaBI (NEB), digested for 1 hour, and heat inactivated.

Publication protocol

"Genomic DNA prepared with the DNeasy purification kit (Qiagen) was
quantified and diluted to 10 ng/l in water. For Il4 methylation analysis,
200 ng of DNA was incubated in enzyme buffer alone or enzyme bufferplus HpaII (NEB) for IL-4 PCR and digested for 1 h followed by heat
inactivation for 20 min at 80°C. Mock-digested DNA and HpaII-digested
DNA were amplified using primers targeting DNase hypersensitive site V
flanking an HpaII site (methylation primers) (forward, 5-GAAGGAGG
AGCAATTTTGCTAATCC-3, reverse, 5-CGTCTTTTCCAGTGAATC
TCTCAAA-3) or control primers lacking an HpaII site from hypersensitive site Va (forward, 5-GAGATGTGAATTCAGGTCCTGA-3, reverse,
5-TGCACACATGCTCTAAATATACAGAT-3) that lie 3 of the IL-4
gene locus. PCR products were detected by ethidium bromide staining after
agarose gel electrophoresis. For Ifng methylation analysis, 84 ng of DNA
was incubated in enzyme buffer with or without 5 U of SnaBI (NEB),
digested for 1 h, and heat inactivated. Mock- and SnaBI-digested DNA
were amplified using 2 SYBR Green Master Mix (Applied Biosystems)
and primers from the promoter (forward, 5-CCCACCTATCTGTCACCA
TCTTAA-3, reverse, 5-CCTCTCTGGCTTCCAGTTTTATACC-3) that
flank a SnaBI site at
53 bp from the transcription start site (methylation
primers) or control primers from exon 1 (forward, 5-GCCAGGCTGCCG
TCTCT-3, reverse, 5-GTCAATAAACACCTCTCCAGCAAA-3) using
real-time PCR. Reactions were quantified against a standard curve of undigested genomic DNA. Fractional methylation is calculated as: quantity of
SnaBI-digested DNA/quantity of mock-digested DNA"

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