We performed DMS footprinting of a 90 nt sequence. The left most lane is A+G ladder. The 2nd and 3rd lane denote DMS reactions in different conditions which are consistent with our hypotheses. The 4th (right most) lane is C+T ladder
We followed Maxam Gilbert PNAS paper protocol for Hydrazine reaction. We used 30 ul of 18M Hydrazine (working concentration), which and mixed in 20 ul of water in ice. After homogeneous mixture is formed we added 5 ul DNA from 10 uM stock (working concentration of DNA 0.9 uM) and immediately incubated in 20 degree for 5 min. Then we stopped the reaction by ice cold sodium acetate (pH=7) followed by addition of 250 ul chilled ethanol and 37.5 ul sodium acetate (pH=5.2) and centrifugation. We did not incubate the sample to avoid salt precipitation Then we followed one alcohol wash with 70% chilled ethanol and piperidine treatment (1M piperidine, 100 uL reaction at 95 degree C for 20 min).
We got the bands with low intensity. More importantly the bands are identical to that of A+G ladder. While Hydrazine is a C+T specific reaction, why does it cleave at A+G and not C+T? How do we optimize the reaction further.
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