NotI / EcoRI digest - (Aug/08/2007 )

I have three inserts of 500bp and 800bp that I want to clone into pSPL3 plasmid. I am going to digest using EcoRI and NotI. My first trial was a double digestion overnight. The PCR products were initially cleaned and checked by gel. After digestion I checked the products again by gel but to my surprise almost everything disappeared. I continued with the ligation and transformation but unfortunately not even a single colony was observed.
Then I decided to do the digestion first using EcoRI followed by NotI. Incubation with EcoRI was done for 3 hrs in EcoRI buffer from NEB. It seems that the same problem is going to happen as the products were really faint following digestion. Now I cleaned from buffer and I am performing an overnight digestion with NotI. I have the feeling that again the experiment is going to fail.
Are there any suggestion about what is happening? Is it due to star activity of EcoRI? I am using 10U enzyme in 50ul reaction (volume of enzyme used is 0.5ul). Is 1 hr enough for digestion?
I was thinking to perform 1hr incubation with EcoRI followed by heat deactivation and then overnight incubation with NotI, is this ok? Or maybe less time for NotI would be fine?
Thanks for suggestions

I think the starting material (initial DNA conc.) is low or You lost a lotof DNA after gel purification.

Check the conditions when star activity occurs (NEB's website). From what you have written, I doesnt seem to occur, but you know better.

Do you have enough bases on the ends of the PCR product for proper digestion. Not1 can be picky about this fact.

Digest the PCR for 2hrs with EcoR1 and then precipitate it then go with the Not1 for 2 hrs also.

good Luck !!!

Hi dnalab,
Never go for the overnight digestion for vector preparation.
It is not recommended.
Niraj

It sounds to me as if you have DNAse or endonuclease contamination of your DNA. The RE buffers and temperature provide a perfect opportunity for these enzymes to work. Where did the DNA come from? What strain? How was it purified? You should be able to digest your DNA in 20-30 minutes tops with these enzymes. You might try to phenol/chloroform extract your DNA, shorten the digestion, or redo the DNA extraction. You should run a lane on your gel of the original and the cut DNA side by side to verify that the DNA is really disappearing, rather than that you had little DNA to begin with. Specs are often inaccurate, and DNA can be chewed up if stored incorrectly.

Thanks for suggestions. I will try with shorter digestion time. There are 12 bases extra to the restriction site. The DNA was of human origin ...extracted from peripheral leucocytes.

I will keep you informed with any modifications.

Might simply be a problem with the water or buffer you use for digestion. Bet you`ve got DNAses in there. Happened to me as well. After 2 h of digestion DNA was totally degraded. Would use another aliquot.