you do not say how much dna you started with Mari or what volume you tried to precipitate but it may be that 5ul simply gives an ok band when whole but after digestion the fragments are smeared over the length of the gel and there is not enough to see. In the same vein when you precipitate 5ul of low concentration dna it may be that there isnot enough dna to precipitate . when precipitating small amounts of dna a co-precipitant like glycogen  ( or rna sometimes) will form a precipitate and bring down the almost colloidal dna with it.

Sorry yes the issue is that when we quantified the rest of the DNA digest sample that wasn't loaded onto the gel,  95ul which had a conc. of about 200ng/ul, there is seemingly no DNA. We're stumped, but the only possibility I could think of is that the DNA degraded whilst I ran the test sample on the gel? 

even if you had severe exonuclease contamination in your DNA sample the fragments and nucleotides would have a significant absorbance and would look like dna using spectrophotometric measurement. What method are you using to quantify dna and what is the od260/280 ratio. I am still concerned that much of your sample is rna which degrades much more easily and will run off the gel very quickly leaving , apparently, nothing. An od260/230 value would also be helpful.

Alternatively If the concentration in the digest is 200ng/ul what was the original stock sample concentration before making up the digestion mixture? I am wondering if the original concentration was very high and the stock solution is very viscous due to the high dna concentration and you failed to pick up the correct volume because the pipette tip was blocked by gloop dna ( pipettes are calibrated on water and not useful for viscous solutions) so ,in fact, no dna was added

Hi Paul, thanks for your help and sorry for the late reply. My mistake it was 200ng/ul starting conc and in the digest we digested 5ug so I believe had a final conc of 0.05ug/ul - hope this helps!

ok I would expect to see 10ng dNA on a gel in a band and you have 50ng/ul or 250ng loaded so if this was smeared over 5 cm of gel then that is 25ng/cm of gel so 10ng would be smeared over 2.5mm of gel. IEven if your sample is all dna ( no rna contamination) this may not be visible...I would run a gel with 20 ul cut dna and a control of uncut dna at 2 concentrations and see if you can see the smear. View the gel at 2 or 3 periods  as the further you run it the weaker the  visible smear of dna will be

There's only one thing I can add to Paul's advice:

Check the digestion pattern with VectorNTI or similar program. The more fragments your reaction should produce, the more DNA you need to load to get a clear gel view. Usually, I digest 1 µg of gDNA in 20 µl, and load 10 µl on the gel. Sometimes that's not enough.

This is very curious. Did you nanodrop an aliquot of the DNA prior to RE digest at the same time as the digested? 

As Paul says, precipitating tiny amounts of DNA is tricky, but glycogen addition works a treat.

Yes the next diagnostic digest we ran we managed to get the smear down. Our sample was measured before the digest fine at 200ng/ul runs on the gel but precipitating it after column or ethanol is giving us very low results. Part of it is probably that it isn't dissolving well as we found when we came back to it the concentrations jumped. Would add glycogen but the DNA is for mouse transfection so unfortunately can't. Is very puzzling.

glycogen is a polymerised sugar Mari. Does it effect transfection?

p

So you are transfecting mouse cells with RE digested genomic DNA? Or is the DNA actually a plasmid? In which case, a smear seems unlikely after RE, unless a combination was used. What is the DNA and what was/were the RE/s? 

My supervisor says glycogen wouldn't be ideal unfortunately, if we really cant get a good concentration then might have to. The digest is to linearise a plasmid with Apa 1 enzyme for recombination into Plasmodium shizonts that are then transfected into new mice. The concentrations for it on the nanodrop are still very low or varied unfortunately after the digest somehow.

I advise doing a maxiprep, or pooling several maxipreps of your plasmid. It seems likely that you would need to repeat the transfections, so starting with a substantial amount of DNA, and scaling up the Apa1 digest would give you lots of aliquots for future use.

Variations in nanodrop readings suggest either the machine needs checking, or more likely, your DNA is heterogeneous. Repeated pipetting through a P200 tip can help to disperse the blobs, then vortex until three separate aliquots of the prep give you the same ND reading +/- 3%.