Can you label the gel? All the same, it looks okay as long the the oligo lengths are correct, MW standards would be helpful. How were the oligos purified?

I could label it but I don't think it will be very helpful. Sizes are ok, upper band is 26 bp and downer is 11 bp. I cleavaged the 26 pb with an enzyme. In the first three all the original 26 bp (almost) is incised and I get only 11 bp. In the last three not all is incised so I should have different intensities of the bands but only 26 bp and 11 bp, not all the background along. I purified with phenol/chloroform and then precipitate the DNA with EtOH/NaAc at -20 oC overnight

Okay, that was helpful. Questions: are the oligos in lane 1-3 the same as lanes 4-6? and was the same restriction enzyme used on oligos in lanes 1-3 as in lanes 4-6? I assume you used less restriction enzyme in lanes 4-6 to see both bands, is that correct?

The smearing in lanes 4/5/6 could be due to several things:

1) the oligos were not purified very well. How were the oligos made? Why did you use phenol/chloroform? That can be used to inactivate an enzyme that was used to make DNA/RNA. Were the oligos purified by the company that made them, and if they were how? If they were HPLC purified then they would be cleaner;

2) the restriction enzyme is either contaminated with a nuclease (if you used a different enzyme for lanes 4-6, since there is not a problem in lanes 1-3), or the oligos have too much salt in them (from the NaAc precipitation) and that is making the restriction enzyme have star activity. Did you cover the precipitated oligo pellet with 70% ethanol for 10 min. at RT to get the salt out before drying them and then re-suspending in TE?

3) the gel was run too fast with too much oligo per well. The bands are very intense so you could load a lot less.

But again, lanes 4-6 do not look that bad, especially if they were not HPLC purified.

1. Cy3 makes the oligos slightly odd to resolve. They do not migrate in the same way as plain oligos. Try 32P labelling as it keeps the native structure.

2. The attachment of Cy3 could be heat-labile, so as it migrates through the gel it gradually breaks down. Try lower voltage. It could also be pH-labile or just labile... hmmm.

3. In general, the gel is overloaded and overexposed. Load a third and expose for a third of the time. Then the relative contribution of the smear will shrink back to what it really is: minimal! :-)

Good luck!

The oligo is always the same, 26 bp, labeled Cy3 in 5' and uracil in position 12. Same enzyme and same amount in all the samples, what changes is the incubation time with the enzyme. But in lanes 1-3 I used NaOH after incubation to cleavege AP sites created by my enzyme and in lanes 4-6 I didn't use. Let's say that lanes 1-3 are controls and I want to check if my enzyme can cleavage the DNA like NaOH does.

I bought the oligos in Sigma-Aldrich, HPLC purification. I used to use 70 % EtOH to clean after precipitation but I lost a lot of signal so I stopped using it and my supervisor suggested using phenol/chloroform and it is a bit better but i still have a lot of backgroud. I also tried to load half of the amount in the gel but the signal decreased quite lot so I had to increase the contrast and the background was the same. the gel is run at 300 V for 2-3 h.

I'm gonna try with 32P radiactivity to see if I get rid of some background and I get better results.

Thanks for the answers, I'll let you know if it goes better :)

Have you run the oligo by itself (no enzyme, no NaOH)? My guess is it would run as a single band. If your enzyme removes purines, but does not cleave the DNA, then from lanes 4-6 it looks like your enzyme has a contaminating nuclease. Did you incubate lanes 4-6 longer than lanes 1-3? Does your enzyme require a metal co-factor?, if it does not need Mg/Mn I would include EDTA in the reaction.

It is ok, the enzyme should for sure excise the uracil (no cleavage, large band) but it seems it can also cleavage the AP site (giving the small band), the only thing is I don't know why all the background in the middle