I had been trying DMS (Dimethyl sulfate) footprinting experiment to elucidate quadruplex folding topology of the putative quadruplex forming sequences.
First time:
The oligos were 6-FAM labelled at 5' end.
1. First annealed in 100 mM KCL as KCL is a stabilizing factor for quadruplexes.
2. PAGE purification.
3. DMS reaction
4. Piperidine mediated cleavage and three wash.
5. Denaturing gel.
Result: I got smeary gel out of which I could not come up to a decision. (Pic: fem3a.JPG)
Second time: I omitted the PAGE elution method, to reduce DNA loss. Rest of the methods are same as above.
Result: The ladder resolved fine but the DMS reaction samples are invisible. I assume they might be lost during the wash. (Pic:IMG-20170502-WA0003 (1).JPG)
I've mostly done RNA footprinting, but I can toss you some ideas.
1. make sure your 70% ethanol for washes after any precipitation is really 70% ethanol. If some of the ethanol has evaporated, it may dissolve more of your DNA. I usually hang on to washes for important experiments in the freezer until I'm sure my stuff isn't in it.
2. I don't know why, but sometimes I've had trouble if my pellet after ethanol precipitation got 'over' dried.
3. Be sure to wash your wells out with buffer again (vigorously) after pre-running right before you run the gel to help your sample run cleanly.
4. Be sure you have enough DMS modification occuring. Are you using cacodylate buffer?
5. You can add something like glycogen as a coprecipitant to help your DNA precipitate and make the pellet easier to see.
6. Consider using formamide loading dye instead of urea loading dye (if you aren't already) as it is more denaturing.
7. If denaturing of DNA might be a problem, you can also heat samples in a heating block (put another heating block on top of the lids to prevent condensation till you're ready to move), then carry the heat block holding your samples to your gel and load directly from the block (like open the tube but leave it in the heating block to pipet).
8. If you have trouble getting a gel without bubbles, I've seen several different ways that you can try to banish them.
Thanks Amanda for your important suggestions.
1. I prefer preparing 70% ethanol prior to the experiment to avoid the probability of evaporation. In this case, I used 1 ml of 70% ethanol for washing. First I added 300 ul ultrapure water then 700 ul directly to the samples. Do you think this might result in DNA loss?
2. I lyophilise the samples after alcohol precipitation. I pour off the alcohol supernatant carefully after which I keep the pellet with remaining alcohol into lyophilization. So, there is always a chance of overdring the sample. Do you prefer air drying instead of lyophilization? If so, what about the downstream steps of footprinting where I must lyophilize the sample?
3. This I will take care in my next trial.
4. I am using 1% DMS dissolved in water (not cacodylate buffer), 5 ul DNA (around 0.5 uM DNA, and 75 ul buffer (100 mM KCL) - total 100 ul reaction volume and the reaction is for about 2 minutes after which I add stop buffer to stop the modification. This is done in most of the papers and earlier I got beautiful results using this same protocol. So, I did not bother to modify this.
5. This is a good idea. In that case, what concentration of glycogen will you recommend to add in the samples and at which step?
6. I use the formamide dye only.
7. I heat the samples into boiling temperature in water bath for 5 minutes followed by snapchilling into ice.
8. I don't have such problems in the denaturing gel.
Please help in this concern.
1. Yes. If you add the water first, it may dissolve some of the DNA. Mix your water and ethanol together before adding to the tube (and make sure no one accidentally bought 190 or 195 proof ethanol instead of 200 proof!).
2. I usually air dry after precipitation, but in later stages I think lyophilization is necessary based on the experimental step.
3. Hope that helps!
4. If water works that is good - if you need to buffer pH then use cacodylate (even though its toxic) as DMS can react with some other buffers.
5. I usually just add a microliter of glycogen (from a company) right before adding ethanol.
6. THat should be your best bet!
7. Try skipping the snap chill and see if it helps. Do whatever makes prettier bands! The heating block is just easier to transport right next to your gel for loading if the water bath is far from your gel apparatus.
8. That's good!
Good luck and let us know how it goes!
It may be worth checking the integrity of your labelled oligos. Oligos stored made up in water absorb CO2 from the air to become acidic and may depurinatr,also too many freeze thaws degrades oligos
Dear Paul,
We have stored the oligo in aliquots. And every time we start with a 10 uM solution. Also, as You can see in the first gel photo, which I have uploaded, there is a high intense continuous smear in the very first lane. This was the ubdigested fraction, do you think it is degraded? If so, I am not supposed to get a resolved ladder which has been prepared out of that sample. Or do you think that the sample is overdigested?
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