[OH-:1].[Na+:2].[CH2:3]([OH:10])[C:4]([NH2:9])([CH2:7][OH:8])[CH2:5][OH:6].Cl>C(O)C(N)(CO)CO.C(N(CC(O)=O)CC(O)=O)CN(CC(O)=O)CC(O)=O>[CH2:3]([OH:10])[C:4]([NH2:9])([CH2:7][OH:8])[CH2:5][OH:6].[OH-:1].[Na+:2]To determine if heating the damaged DNA in the presence of salt can improve the amplification of this substrate, damaged DNA (25 ngs) was heated to 70° C. in the presence of a salt (Tris-EDTA (10 mM Tris pH 8.0 and 1 mM EDTA)) for 3 minutes, 25 ngs of native damaged sample was added back, and then the sample was cooled. This method showed some improvement on the repair of the damaged DNA. When 25 mM final of NaOH was added to the reaction before the heat step, this improved the repair method giving consistent repair of the damaged DNA. This was done by diluting damaged DNA in Tris-EDTA (TE) to 5 ng/μl, 25 ngs of DNA was added to NaOH final concentration 25 mM, the sample was heated to 70° C. for 3 minutes, Tris-HCl pH 4.0 was added to reduce the pH to 8.0, 25 ngs of the original native damaged DNA sample was added back, and the mixture was cooled to room temperature. Repair with heat, Tris, and NaOH gave better locus representation than the no repair and repair with heat and Tris only treatments.
Wird geladen…
