restriction enzyme digestion problem - (May/22/2008 )
I tried to digest my BAC DNA using the NotI (NEB) for sizing but the digestion patterns came out different everytime when I digest under same concentration and condition. I got a ~16kb size BAC under 4h digestion and ~35kb size BAC under 16h digestion. Then I tried with other different enzymes (BamHI, EcoRI, HindIII, SmaI...), non of them gave me a consistent banding pattern after electrophoresis. The digestion condition was same for every enzymes but I just could not get the same banding pattern everytime. I tried to change the new enzyme, buffer, BSA, water also came out with different pattern. 
I followed exactly what the supplier recommended condition to avoid star activity and overdigestion still cannot get the same pattern. Was it my BAC problem or diegstion problem? 
Thanks!
A common problem is too high a DNA concentration. What volume and amount of DNA are you using? How much enzyme are you adding?
I put in 500ng of BAC DNA, tried with different unit of enzyme (5U, 10U, 15U, 20U), all get the different bands also.
I hope you are using the same BAC preparation everytime. BACs are prone to rearrangement.
I always use the QIAGEN Large Constrcut Kit to extract the BAC DNA. I run gel and read nanodrop to identify the BAC quality and quantity then only digest it.
I always use the QIAGEN Large Constrcut Kit to extract the BAC DNA. I run gel and read nanodrop to identify the BAC quality and quantity then only digest it.
whichever method you use, make sure that in all digest comparsions you are making, you have used a BAC DNA that have all come from a single colony on a plate. Not from many different colonies, nor from a colony that has been propagated many times over in between DNA preps.
This is generally not a big problem, but if your particular BAC has a lot of rearrangment prone sequence, you may get subtle or dramatic changes in restriction pattern. So, culture a colony, make a mega prep, fingerprint it extensively to make sure that there is no rearrangement and that you are getting all bands right (you may have to do a southern, not just digest), and then use that same DNA for all your expts.
If you run out of DNA, do the same procedure all over again, don't think that your BAC sequence will be the same.
You didn't answer my question about the volume of the reaction you are doing. It makes a big difference if you have 500 ng in 10 ul or in 100 ul. Also, the fraction of the volume which is enzyme can also be a problem.
We usually prepare the digestion reaction in around 20ul - 30ul. The other enzymes seems not giving any problems now, just the NotI is still not consistent when the incubating time is different (16h and 4h). According to NEB, NotI should not have star activity but what we seen from gel, the pattern was just like star activity. Thanks!
I would do this reaction in at least a 50 ul volume. High DNA concentrations lead to star activity, as does high glycerol concentrations resulting from adding the (50% glycerol) enzyme to a low volume reaction. I don't know if this would be the case with NotI -- I avoid this problem by never trying.