Help! my "plasmid" won't cut. - RE digestion problem (Jan/09/2007 )
Ok. Ive been doing mol bio for >10yrs and never come across this. eporate cosmid, plate, select colonies, grow cultures, do Q midi prep. spec is great--260/280 & 260/230. cant digest with bamh1 (looks same as uncut). cant digest with ecor1. the prep that i used to eporate cuts each time. phenol extraction doesnt help. overnight, 24, 48 and 72 hr digestions dont work. overdigestion doesnt work.
uncut looks more or less as expected usually 3 bands (sometimes more) with the lowest being most intense (supercoiled-running at about 5kb because 45kb cosmid).
im mad enough that i cant get usable cosmid dna, but now i want to know why i cant cut this dna. even if it the cosmid is unstable and had grossly rearranged/deleted then it should still cut with bam or eco (each have about 10-15 sites in cosmid). its not chromosomal contamination. my only guess is its contaminating plasmid that has amp marker too but has no eco or bam sites. seems pretty unlikely.
this isnt a one time event. it has happend with each of 5 cosmid clones of a viral library multiple times. i also thought about methylation, but bam and eco are more or less resistant.
help, i need more cosmid dna. 
maybe the enzyme's are off.
V
Here are my two cents
could the enzymes be dead? Can it be confirmed that the enzymes still work
could there be a denatured DNA problem... an unusually severe one? Different growth conditions can make the cell walls stronger or weaker... thus altering the proper lysis time.
could there be severe contamination? Was the electroporation cosmid extracted from the same strain as the one you are using now? Can you use the strain used to make the electroporation cosmid? What e coli strain are you using?
Too much DNA can prevent digestion by restiction enzymes
increase the reaction volume and add much more enzyme 
V
nah, the cosmid prep that i used to eporate to begin with cuts great every time.
could the enzymes be dead? Can it be confirmed that the enzymes still work
could there be a denatured DNA problem... an unusually severe one? Different growth conditions can make the cell walls stronger or weaker... thus altering the proper lysis time.
could there be severe contamination? Was the electroporation cosmid extracted from the same strain as the one you are using now? Can you use the strain used to make the electroporation cosmid? What e coli strain are you using?
Too much DNA can prevent digestion by restiction enzymes
dont know about denatured dna possibility. i am getting multiple bands when i run uncut. usually only 2 or 3 but some times more. what would denatured dna look like?
yes, i used the same coli strain that was initially used dh10b. also subsequently used dh5alpha.
think i might try a 4base cutter. that should chew up this mystery dna
also might just eporate some control plasmid to make sure i can actually prep dna that will cut, but it wont be a cosmid or low copy cuz we dont have any.
guess i could try that, but im only cutting 2 ug in 100ul. besides, its not like im getting partial digestion.
any chance you could sequence the plasmid...
ohhhh just thinking, i know someone who is in the process of making a viral DNA library, and when she was checking the itial cloning proces, she had contamination with an other plasmid that ran just slightly above the plasmid she needed. i'm pretty sure the contaminating plasmid didn't cut with the enzyme she needed as well. i think she had to go right back to the beginning (sorry).
cut with a whole bunch of RE and see what happens.
V