Smear in my restriction digest - (May/07/2009 )

Hi I was just wondering if anyone could help me. Whenever I do a restriction digest all i get is a big smear. I have tried multiple different digests and they all result in a smear. To check for DNase I contamination i incubated my pDNA with water and the pDNA was not degraded. I then also incubated my pDNA with water and buffer and this results in a smear which suggests the contamination is in the buffer. I have tried the digests with many different buffers and all result in a smear. I have tried heating the buffer and pDNA at 75oC for 10mins to inactivate any DNase but it still results in a smear. I was wondering if anyone had any suggestions. Also is it possible that there is something else degrading my DNA other than DNase?

Thanks in advance for any help

panda on May 7 2009, 03:43 AM said:

Hi could you tell us the nature of your DNA, is it genomic DNA or plasmid DNA.

If the problem is DNAse, use phenol chloroform to deactivate the enzyme.

You should change all your reagents, (buffers, BSA, water) with new reagents. THat would eliminate the potential that the contamination.

panda on May 7 2009, 01:43 PM said:

I strongly believe that the smear is most probably due to Dnase in your plasmid prep and not the buffer because:

1. DNases need Mg++ for activity and thus it is not surprising that incubation of plasmid in water does not give a smear.

2. When you supply Mg to plasmid+water you get a smear. Also, you get a smear with different buffers. They cannot all be contaminated with DNase!

3. Inactivation of DNase by heat is tricky. Inactivation is dependent on Mg concentration and "inactivated" enzyme can regain activity on addition of Mg++ (see ref). Also remember that E. coli probably contains DNases that are more heat-stable than the commonly used reagent DNase 1.

Is the E.coli strain you maintain the plasmid in endA- ? These strains usually result in better plasmid preps (less degradation). Check the genotype and if not transform plasmid into a common endA- strain like DH5a, TOP10 etc.
I certainly agree that a phenol extraction could help. But using plasmids with a prior history of DNase exposure is always a worry. Better the make a new stable maxiprep.

See:

HANAKI K et al. DNase I activity retained after heat inactivation in standard buffer.(2000) BioTechniques 29: 38-42

Hope this helps

Thanks for your help. I am using pDNA and I am transforming it into DH5a cells. I suspect that the pDNA may be getting contaminated during the pDNA prep so I was thinking the best idea might be to do the suggested phenol/chloroform extraction. I was wondering if anyone had good protocol for doing this on my current pDNA?

Thanks again

panda on May 8 2009, 12:48 AM said:

What volume is your DNA sample in?

The general thing to do is to add a volume of phenol/chloroform/isoamyl alcohol 25:24:1 (equilibrated to pH8) to 5 volumes of your DNA solution. So phenol/chloroform : DNA solution = 1:5

Mix the two. Then spin it down using a centrifuge approximately 2min at maximum rpm. Then move the aqeous supernatant to a new tube. Repeat the phenol/chloroform extraction until the interphase between the organic and aqeous layer is clear.

Once you have the aqeous solution add 3M of Na acetate (1/10 volume). Then add 3 volumes of 100% ethanol. Then cetrifuge at maximum rpm for 10min.

The DNA pellet should be on the wall of the tube. Discard the supernatant. Wash pellet with 70% ethanol (you can spin the DNA pellet to prevent it from detaching from the wall of the tube. Discard the wash and briefly dry your DNA (do not over dry), then resuspend the DNA in TE.

Take a bit of your plasmid prep, and incubate at 37 for an hour, then run it on a gel. If you still have plasmid, I doubt you have DNase. If you have an empty gel, sorry but you will have to clean up as pernesblue said, or better still start again.

I had the same problem...for me the solution was very simple...cleaning the gel chamber and fresh running buffer solved the problem. It could however also be a problem of the loading buffer...don't forget to consider this options, when having a problem like this...also using filtered tips could help...

ooolex on Jun 19 2009, 04:18 PM said:

Hmm... the loading buffer was a good point. The buffer gets contaminated pretty easily, especially if a lot of people are using it.

deactivate DNase with EGTA

I have met the same problem. Now I have solved it successfuly. It is very easy, just transfer you pDNA into Top10 or DH5a wich are end- strains