werner@aecom.YU.EDU (Craig Werner) (07/03/87)
The following hint came about after I was consistently able to get 10 times as much plasmid (pUC18) DNA from a 1.5 ml (Eppendorf tube) miniprep than any one else on our floor. I was averaging about 20 micrograms per tube - 1/500 of the prep was visible after after digestion, whereas others were digesting half or all of the prep to see bands. What did I do differently? Everybody else used old cultures or cultures put on a roller, or just in the 37C incubator overnight. I started instead doing the following: Placing 5 ml of NZYM into a 250 ml Erlemeyer flask and inoculating that, letting it shake vigorously overnight, and doing the plasmid prep first thing in the morning. I credit it all to aeration, although like everybody else in biology, I changed a few steps later in the protocol as well. Also, concerning gels: normally 1 mg/ml Ethidium Bromide is used as 2000X solution (i.e. 50 ml gels get 25 ul EthBr). I've been using 12 ul for years (4000X) with no adverse results, and I just went down to 8 ul (6000X) ditto. L prefer it because you don't see the background haze at the top of the gel. The fact that it also throws less carcinogens around lab is also a positive point. -- Craig "Baby Doc" Werner (future MD/PhD, 3 years down, 4 to go) werner@aecom.YU.EDU -- Albert Einstein College of Medicine (1935-14E Eastchester Rd., Bronx NY 10461, 212-931-2517) "Time flies when you're streaking out N. gonorrheae."
lonetto@phri.UUCP (Michael Lonetto) (07/11/87)
Along the same lines as Craig's tips on mini preps: we have often had to screen >>100 transformant lines (my record was 400)for proper restriction pattern, which puts a great deal of strain on shaker space and glassware if you use flasks. Our answer was to use "potatoe tubes", 25x175 mm glass culture tubes, with 1-2 ml of media, and shake vigorously (300-350rpm) overnight. One critical variable is the length of incubation: Too long and you're sunk (13-15 hrs worked well for 2 ml cultures of JM105/pUC). Another was the efficiency of lysis (make up the lysozyme fresh). Working this way I was able to determine restriction maps using 0.5-1% of the yield from 1 ml of culture (one miniprep gives plenty of material to purify a fragment and reclone it, a great timesaver). -- Michael Lonetto UUCP:(allegra!phri!lonetto) USMAIL: Public Health Research Institute, 455 1st Ave, NY, NY 10016