DNA smears for the last 5 weeks!!!

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habbas
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DNA smears for the last 5 weeks!!!

Post by habbas » Mon Nov 26, 2007 12:25 am

Hi;

I really hope anyone can offer some help or his experience with this.
For the last 5 weeks, I keep getting smears when I cut my vectors. I used vectors that worked few months ago but when I cut them now with different enzymes (more than 10 different options) and different buffers, I still get smears. I even borrowed enzymes from other labs, changed buffers, changed water, changed BSA and I changed tubes. I cleaned everything around but still I am gettin degradation. The uncut seems so good even when I incubate it with no enyzmes at 37. However, when I add everything without the enzyme, I get smears as well.. so It could be a nuclease problem that is acticated by the buffers. But what's about the old vectors that I used before and worked perfectly, why are they also giving this kind of degradation now? It is so annoying and I really cant fix it. I used Qiagen miniprep kits and I retransformed and replated yet I am still getting the problems. I chagned water as well. For only one week during the last 5 weeks things worked perfectly then for reason I dont know of things got worse again. Anyone can give me his feedback on this please?

I really appreciate any kind of suggestions and help.
Last edited by habbas on Mon Nov 26, 2007 1:31 am, edited 2 times in total.

habbas
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Post by habbas » Mon Nov 26, 2007 1:31 am

I wanna add one thing.
I just did the same reaction along with my friend. The only difference was that she used different water however nad we incubated 2 hours on different incubators and we still got the degradation at 37.
Last week's cuts were just so good. It all came back again.. this is crazy!

By the way, if I boil my plasmids at 100 degrees then I let it cool, would that disrupt it? The idea is I want to kill the nucleases if any. Please let me know.

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Post by ddoran » Sun Dec 02, 2007 4:28 am

Even if there are nucleases, the enzyme will still renature once the denaturation factor is removed from the solution. I can't really tell you what would happen to the plasmids if they are boiled, however.

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canalon
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Post by canalon » Mon Dec 03, 2007 12:50 am

Boilng should be OK, but as ddoran said it might not get rid of all nuclease, although DNAse are not the most stable enzymes. Maybe your vector are degraded (you might have checked, but since you do not say...) or your purification procedure is not OK
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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Post by -snook- » Mon Dec 03, 2007 3:14 am

maybe protein contamination in your prep
idk?

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