Nco1 digest problem - (Mar/25/2010 )
Hello, I am screening colonies for an insert by digestion with Nco1. My plasmid preps have yield ~.3 ug/ul. For each reaction I have: 4 ul plasmid, 1ul #3 buffer, .2 ul Nco1, 4.8 ul H20 for a total of 10ul. I digest for ~2 hours at 37 C. When I run my samples on the gel I just get a smear of DNA in the 100 bp to 500 bp range. I do expect a band at 300 bp but also a 1kb band and 4 kb band. I also have checked my plasmid on a gel and analyzed DNA content spectroscopically. Why am I getting this smear?
First up, I'd try a different lot of NcoI. If you get the same problem, try a different enzyme. If that still gives a smear, try reprecipitating the DNA. I just hope you have enough DNA for all of these tests...
are you using 0.2Ál enzyme? then maybe try to use less DNA like 250ng. If you are using 2Ál increase the rxn volume to at least 20Ál.
I think you had a very high enzyme:DNA ratio. This could lead to the star activity. To overcome that, you can add the total volume to 20ul, but since the DNA amount is very low, you need to increase the DNA also. I suggest to use 10ul plasmid DNA, 2ul buffer, .1ul NcoI, 7.9ul H2O and incubate less than 2h. Also, another problem you may get is the endogenous enzymes of the host (endA+ strain) if you used the wild type strain, in this case, you should add one buffer in the midipreps kit to exclude the enzymes.
The fraction of your digestion reaction that is composed of your DNA solution is very high. Any contaminants in your DNA can easily inhibit the reaction. I would do this reaction in 50 ul volume, with 4 ul DNA, 1 ul RE, 5 ul buffer, 40 ul water. In this reaction, 80% of the volume is water, with no impurities. The lower concentration of enzyme (and hence also lower concentration of glycerol) also helps. This is what your enzyme manufacturer recommends, also, I might add. I would probably do this with NcoI-HF, and switching to buffer 4.