This is a very interesting topic I have to say. But what I missed in this discussion is the pain you go through when freezing in the cold room. As the name implies it's supposed to be cold (most of the times). But that's not too much of an issue as you can dress up accordingly. The problem I always had was freezing up of the <advertisement> Hampton Magnetic Wand </advertisement> and icing up towards your fingertips after some time when moisture from the cold room condenses and freezes. I hate wearing gloves when handling crystals so there was not much of a skin protection.
How do you guys solve this problem ? Jürgen ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Jul 13, 2012, at 8:01 PM, <mjvdwo...@netscape.net<mailto:mjvdwo...@netscape.net>> <mjvdwo...@netscape.net<mailto:mjvdwo...@netscape.net>> wrote: FYI, I live at 5500 ft elevation and the oxygen content of air is 21.5% here. The TOTAL amount of oxygen is less where I am than where you are because there is less air (lower density). Therefore my body has to do more work to get the same amount of oxygen to my cells. OSHA has nothing to do with how much air we have and the sensors you can buy will tell you that the percentage is 21.5, even at 5500 feet. No, nitrogen is not toxic. The question is if you can displace enough oxygen so you cannot absorb enough of it anymore. Unlike the experience you may have had when you hold year breath, you can breathe fine, there is lots of gas around you. Just not the right kind. We are not on the cusp of suffocating. On the other hand, paradoxically, oxygen is toxic. When you get too much of it, you will damage your CNS (not the program) and your eyes. When premature babies are given oxygen so they can survive, their eyes may get damaged. Too far removed from CCP4. This cannot happen in the cold room while harvesting crystals. Mark -----Original Message----- From: Jacob Keller <j-kell...@fsm.northwestern.edu<mailto:j-kell...@fsm.northwestern.edu>> To: mjvdwoerd <mjvdwo...@netscape.net<mailto:mjvdwo...@netscape.net>> Cc: CCP4BB <CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk>> Sent: Fri, Jul 13, 2012 5:10 pm Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal) The expansion ratio of liquid to gaseous nitrogen is approximately 1:700, that is, 1 liter of liquid becomes 700 liters of gas (at room temperature). When you are in a room that is 3 (~10ft) meters tall, 6 (~18ft) meters wide and 10 (~30ft) meters long and you assume that it is poorly ventilated (i.e. no gas replacement at all), then you will have 3x6x10 = 180m3 volume of gas, which is 180,000 liters. Air consists of 21% oxygen and is considered deficient if it goes down to 19.5%. OSHA recommends having monitors present in the case you might, in worst case scenario, reach 19.5%. Note: I don't know, but it seems unlikely that you are critically injured at 19.5% How can this OSHA number be right? At fairly high altitude, say 2500 m, the partial pressure of O2 will be about 75% of that at sea level, and most are okay with it--so how can a drop from 21% to 19.5% have any importance? Is N2 competing with O2, perhaps? Never heard of that. Can N2 really be a poison, such that we are constantly poised at the cusp of suffocation? JPK In this hypothetical case, you will have about 37800 liters of oxygen. If you displace some of it with 700 liters of nitrogen (you spilled one liter of liquid nitrogen), you will be down to 37100 liters, or approximately 20.5%. So, no worry. If you have cryogenic storage for crystals (typically hundreds of liters) or one of those large tanks to back-fill your cryo-system, the story changes a lot. Large dewars or large tanks for filling do not normally fail, but when they do, you will be at risk. Humans cannot sense the lack of oxygen, you just feel sleepy and keel over. So in small rooms with large amounts of liquid nitrogen, it makes sense to have a monitor (and it does not make sense to be scared of the issue when you have a monitor). Educationally: For each safety risk in your environment you are supposed to do a calculation like the one above and consider how likely (or not) it is that this may happen to you and how bad it will be. Likelihood and severity multiply: if it is very unlikely (that a large nitrogen tank will rupture) but the consequence is severe (you die), then you need to think about how you can make sure that it never happens (install sensor). Conclusion: if you only work with a small open dewar, then even in a small room it is highly unlikely to run out of oxygen. It is an excellent idea to ask questions like you did. It should be expected that your institution has experts who can answer such questions, but some (like ours) do not and you have to figure it out yourself. It is a good idea to document your concern, calculation and recommendation. Hope this helps. Mark PS: nirtogen vendors have excellent reference materials about these things. -----Original Message----- From: Radisky, Evette S., Ph.D., Ph.D. <radisky.eve...@mayo.edu<mailto:radisky.eve...@mayo.edu>> To: CCP4BB <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> Sent: Fri, Jul 13, 2012 3:19 pm Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal) Several have mentioned harvesting in the cold room to reduce evaporation. I used to do this also as a postdoc, but I worried whether I risked nitrogen gas poisoning from liquid N2 boil-off, since the cold room did not seem very well-ventilated. I’ve also hesitated to recommend it to trainees in my current lab for the same reason. Does anyone have solid information on this? I would like to be convinced that such fears are unfounded … Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK?>] On Behalf Of Roger Rowlett Sent: Thursday, July 12, 2012 2:11 PM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] cryo for high salt crystal We frequently crystallize one of our proteins and variants of it in 1.6-1.8 M ammonium sulfate solutions. Cryoprotection with 25-30% glycerol or 25-30% glucose does not cause precipitation of salts. Both KCl (4.6 M) and ammonium sulfate (5.6 M) have enormous solubilities in water, so I would not expect cryoprotectant concentrations of glycerol or glucose to cause precipitation (We can save cryoprotectant solutions of at least 2 M ammonium sulfate indefinitely). How are you introducing cryprotectant? We use one of two methods: 1. Fish the crystal out of the mother liquor and place into artificial mother liquor with the same composition as the well solution + cryoprotectant. For glycerol or other liquids, you have to make this from scratch. For glucose, we just weigh out 300 mg of glucose in a microcentrifuge tube and make to the 1.0 mL mark with well solution. (Mix well of course before use. Gentle heating in a block or sonication will help dissolve the glucose. 2. Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to the drop the crystals are in. You can do this all at once, or in stages, keeping the drop hydrated by placing the hanging drop back in the well between additions. If your drops are drying out during crystal harvesting (very possible in dry conditions), you might try harvesting in the cold room, where evaporation is slower. We often have problems with crystal cracking and drop-drying in the winter months when the humidity is very low indoors. The cold room is usually humid enough and cold enough to slow evaporation to allow crystal harvesting. (I hate working in the meat locker, though.) Cheers, _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu<mailto:rrowl...@colgate.edu> On 7/12/2012 12:55 PM, m zhang wrote: Hi Jim, 25% is w/v. Thanks for the information. Will check the webinar. Thanks, Min ________________________________ From: jim.pflugr...@rigaku.com<mailto:jim.pflugr...@rigaku.com> To: mzhang...@hotmail.com<mailto:mzhang...@hotmail.com>; CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: RE: [ccp4bb] cryo for high salt crystal Date: Tue, 10 Jul 2012 17:39:56 +0000 Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v or v/w?), try using 100% saturated in reservoir, 75% saturated in reservoir, or 50% saturated in reservoir. You will have to TEST these. See also this webinar on cryocrystallography which shows how to make these solutions: http://www.rigaku.com/node/1388 You could also try high salt solutions with similar technique. Good luck! Jim ________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] on behalf of m zhang [mzhang...@hotmail.com<mailto:mzhang...@hotmail.com>] Sent: Tuesday, July 10, 2012 11:28 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] cryo for high salt crystal regaentDear All, I am sure this question was discussed before. But I am wondering if anyone got the same experience as I do. I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or ammonium sulfate itself: The problem is that all the cryo plus original reagents in the reservoir precipitate the salts out. And more serious problem is because of high salt in the condition, while I am trying to loop the crystal, both the drop and cryoprotectant drop form salt crystals (not sure it is KCl or ammonia sulfate) significantly and very quickly, that cause my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does anyone here have similiar case? any suggestion will be appreciated. Thanks, Min -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu<mailto:j-kell...@northwestern.edu> *******************************************
