User:Torsten Waldminghaus/Notebook/RNA methylation by dam: Difference between revisions

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(New page: ==Oligos== oligos for methylation experiment from <cite> Humeny-2003</cite>: GATC19DNAfw GCCCGCGGATCCGCCCGCC GATC19DNArv GGCGGGCGGATCCGCGGGC GATC19RNAfw GCCCGCGGAUCCGCCCGCC GATC19RNA...)
 
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==Reaction==
==Reaction==


100 μL:
*for 100 μL:
*4μL primer fw (100pmol/μL = 100μM) ~2.4 μg
**4 μL primer fw (100pmol/μL = 100μM) ~2.4 μg
*4μL primer rv (100pmol/μL = 100μM) ~2.4 μg
**4 μL primer rv (100pmol/μL = 100μM) ~2.4 μg
*2μL Dam (NEB) (8U/μL) =16U
**10 μL 10x Dam buffer (NEB)
*   S-adenosylmethionine (SAM) 80 μM
**80 μL A. dest
*10μL 10x Dam buffer (NEB)
=> for primer annealing boil about 800 ml water add reaction tubes with the above content and store at room temperature until the water cooled down to room temperature (or at least 37 °C
*add the following:
**2 μL Dam (NEB) (8U/μL) = 16U
**0.5 μL S-adenosylmethionine (SAM) (32mM) = 160 μM


=> Incubate for 1h at 37 °C
=> Incubate for 1h at 37 °C


*Add 100 μL A. dest and extract with phenol-chloroform  
*Add 100 μL A. dest and extract with 1 x phenol and 1x chloroform  
*Ethanol precipitation
*Ethanol precipitation


*'''1X dam Methyltransferase Reaction Buffer (NEB)''':
**50 mM Tris-HCl
**5 mM 2-Mercaptoethanol
**10 mM EDTA
**pH 7.5 @ 25°C




Revision as of 04:30, 6 May 2008

Oligos

oligos for methylation experiment from [1]:

GATC19DNAfw GCCCGCGGATCCGCCCGCC

GATC19DNArv GGCGGGCGGATCCGCGGGC

GATC19RNAfw GCCCGCGGAUCCGCCCGCC

GATC19RNArv GGCGGGCGGAUCCGCGGGC

Reaction

  • for 100 μL:
    • 4 μL primer fw (100pmol/μL = 100μM) ~2.4 μg
    • 4 μL primer rv (100pmol/μL = 100μM) ~2.4 μg
    • 10 μL 10x Dam buffer (NEB)
    • 80 μL A. dest

=> for primer annealing boil about 800 ml water add reaction tubes with the above content and store at room temperature until the water cooled down to room temperature (or at least 37 °C

  • add the following:
    • 2 μL Dam (NEB) (8U/μL) = 16U
    • 0.5 μL S-adenosylmethionine (SAM) (32mM) = 160 μM

=> Incubate for 1h at 37 °C

  • Add 100 μL A. dest and extract with 1 x phenol and 1x chloroform
  • Ethanol precipitation


  • 1X dam Methyltransferase Reaction Buffer (NEB):
    • 50 mM Tris-HCl
    • 5 mM 2-Mercaptoethanol
    • 10 mM EDTA
    • pH 7.5 @ 25°C


  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 | PubMed ID:12576072 | HubMed [Humeny-2003]
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