User:Torsten Waldminghaus/Notebook/RNA methylation by dam

Oligos

oligos for methylation experiment from [1]:

GATC19DNAfw GCCCGCGGATCCGCCCGCC

GATC19DNArv GGCGGGCGGATCCGCGGGC

GATC19RNAfw GCCCGCGGAUCCGCCCGCC

GATC19RNArv GGCGGGCGGAUCCGCGGGC

Reaction

100 μL:

• 4μL primer fw (100pmol/μL = 100μM) ~2.4 μg
• 4μL primer rv (100pmol/μL = 100μM) ~2.4 μg
• 2μL Dam (NEB) (8U/μL) =16U
• S-adenosylmethionine (SAM) 80 μM
• 10μL 10x Dam buffer (NEB)

=> Incubate for 1h at 37 °C

• Add 100 μL A. dest and extract with phenol-chloroform
• Ethanol precipitation

1. Humeny A, Beck C, Becker CM, and Jeltsch A. Detection and analysis of enzymatic DNA methylation of oligonucleotide substrates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Anal Biochem. 2003 Feb 1;313(1):160-6. DOI:10.1016/s0003-2697(02)00568-7 |