My cold room is also too small for comfort, but I think your method could work in a pinch. Thanks!
Evette Radisky, PhD Mayo Clinic Cancer Center Griffin Cancer Research Building 4500 San Pablo Road Jacksonville, FL 32224 tel: 904-953-6372 fax: 904-953-0277 On Jul 13, 2012, at 5:52 PM, "tom.p...@csiro.au" <tom.p...@csiro.au> wrote: > Hello Evette, > > It will depend on the circumstances- how big is your cold room, how much > liquid N2 you have in there, etc. > You can actually calculate the percentage of N2 (or correspondingly, O2) in > the air if all of the liquid N2 were to boil off at once. > If the O2 goes below 20% it will feel like you are at elevation and if it > goes below 19% it isn't good (I believe most oxygen sensors used for this > kind of application alarm if it goes below 20% and then alarm strongly below > 19%). > As you, we generally avoid cryo-cooling crystals in the cold as we have a > small cold room and no real ventilation- just a blower. > If we use liquid N2 in there, we keep the door open and have someone stand > outside. There is no warning with N2- you just fall unconscious. > > Best of luck, tom > > Tom Peat > Biophysics Group > CSIRO, CMSE > 343 Royal Parade > Parkville, VIC, 3052 > +613 9662 7304 > +614 57 539 419 > tom.p...@csiro.au > ________________________________________ > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Radisky, > Evette S., Ph.D. [radisky.eve...@mayo.edu] > Sent: Saturday, July 14, 2012 7:19 AM > To: CCP4BB@JISCMAIL.AC.UK > 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] On Behalf Of Roger > Rowlett > Sent: Thursday, July 12, 2012 2:11 PM > To: 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 >
