Looking for a protocol for a partial digest w/ NdeI - (Apr/09/2007 )
I have a vector ~6000 bp in size that has my DNA fragment of interest between NdeI and XbaI sites. So it has one XbaI site but two NdeI sites, the second one on the vector.
To remove my fragment of interest I plan to do a complete digest w/ XbaI followed by a partial digest w/ NdeI at different timepoints and then run on a 2% gel. The fragment I need is ~500 bp.
I am looking for a good protocol that will allow me to do the partial digest.
I came across 2 options -
1) after addition of enzyme, incubate at 37degC and remove at different timepoints and keep on 10X icecold LB? and then run on a gel.
I want to know if I would need to keep it on LB coz I am not growing anything.
2) Adding EtdBr to my reaction at different timepoints - but I did not find any definitive protocol for this - amounts of stuff??
Can anyone direct me to a protocol to do this kinda digest?
Any help is appreciated.
NEB's catalog states that standard miniprep DNA is cleaved more slowly by Nde I.
Many years ago, I found that I could frequently alter the site preference for partial digests by using DNA prepared by 4 methods:
1) standard miniprep without phenol/chloroform extraction,
2) standard miniprep with phenol/chloroform extraction,
3) cesium gradient purified DNA without phenol/chloroform extraction, and
4) cesium gradient purified DNA followed by phenol/chloroform extraction.
You may also see site preference variability between supercoiled and linear DNA, so try Nde I before and after your Xba I cut.
Ethidium bromide causes photo-oxidation of DNA with visible light (not just UV lamps) and molecular oxygen. So restriction cuts in the presence of ethidium bromide should be done in the dark. Unfortunately, I used this technique about 15-20 years ago and no longer remember what concentration of EtBr was used.
I suspect in your note, the 10xLB should be 10X TBE instead.
The other way to do so is titration of enzyme that you use vs DNA amount.
You expect to see a mixture of partial digestion and fully digested fragments
and you calculate all possible combinantions to decide which band to pick.
My question to you is: suppose protein-DNA complexes are the reason behind this selectivity that you have noticed, is that due to the fact that proteins binds selectively to a specific region in the plasmid in bacteria? Otherwise the reduced cutting would have been random event and you would not see the selectivity.