We have recently performed DMS (dimethyl sulphate) footprinting experiment to understand the folding topology of the quadruplex forming sequence (90 nt). The sequence is labelled with 6-FAM at its 5'. The resolution of the A+G ladder is still better, but the sample (quadruplex motif) looks like a smear and did not resolve properly. Our protocol is explicitly divided into four steps: 1. Annealing (to allow quadruplex formation), 2. PAGE purification, 3. DMS reaction and piperidine cleavage, and 4. denaturing PAGE (10%)
1. Annealing (to allow quadruplex formation), 2. PAGE purification, 3. DMS reaction and piperidine cleavage, and 4. denaturing PAGE (10%)
2. PAGE purification (10% native PAGE)
3. DMS reaction and piperidine cleavage: We used 1% DMS for 2 minutes reaction and 1 M 10% v/v piperidine (fresh) for 20 minutes at 95 degree.
4. denaturing PAGE (10%): We did not measure the concentration of the DNA, we loaded because nanodrop measurement might be erroneous because of the interference of the single stranded cleaved sequences. We started with 10 uM DNA, however, we lost DNA at two successive alcohol precipitation steps. Finally dissolved in 10 ul formamide dye and loaded.
In my opinion is done all the steps correctly.
Focus on set-up devices and Temperatures
Here is the next trial of footprinting. Resolution improved than before, but now only the ladder is visible the samples are either smear or invisible. The difference from the the last time is I have omitted the PAGE purification step. Rest of the method remains the same.
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