Hello Megha, My first thought also was contamination (how does your culture look on a plate or under the microscope?), hadn’t thought of cell lysis. Another possibility - perhaps your strain has acquired a mutation that allows it to better tolerate the protein overproduction?
For either the mutation scenario or contamination, can you go back and reintroduce the gene in the strain, see if this happens again? Barbara From: Wolfgang Schechinger <hubah...@gmx.de<mailto:hubah...@gmx.de>> Subject: Re: Slimy recombinant e.coli pellet Date: 9 November 2017 10:13:58 GMT-5 To: Megha Goyal <mgbio...@gmail.com<mailto:mgbio...@gmail.com>>, meth...@magpie.bio.indiana.edu<mailto:meth...@magpie.bio.indiana.edu> Looks like contamination, Megha. Do you find your protein in the slime? Regards, Wo On 09.11.2017 14:11, Megha Goyal wrote: Dear All, We are doing fermentation for recombinant protein using E.coli culture. Earlier we used to obtain compact cell pellet on harvesting (3 Hrs after IPTG Induction). But recently we observed that the cell pellet harvested are of slimy consistency and they do not homogenise in the washing buffer (tris-nacl-edta). We observe thread like consistency and no matter of stirring it do we get a homogeneous suspension. Can someone guide me what is the reason for such phenomenon. Thanks megha _______________________________________________ Methods mailing list Methods@net.bio.net<mailto:Methods@net.bio.net> http://www.bio.net/biomail/listinfo/methods From: d...@no.email.thankstospam.net<mailto:d...@no.email.thankstospam.net> (DK) Subject: Re: Slimy recombinant e.coli pellet Date: 9 November 2017 19:21:24 GMT-5 To: methods@net.bio.net<mailto:methods@net.bio.net> In article <mailman.110.1510233337.15473.meth...@net.bio.net<mailto:mailman.110.1510233337.15473.meth...@net.bio.net>>, Megha Goyal <mgbio...@gmail.com<mailto:mgbio...@gmail.com>> wrote: Dear All, We are doing fermentation for recombinant protein using E.coli culture. Earlier we used to obtain compact cell pellet on harvesting (3 Hrs after IPTG Induction). But recently we observed that the cell pellet harvested are of slimy consistency and they do not homogenise in the washing buffer (tris-nacl-edta). We observe thread like consistency and no matter of stirring it do we get a homogeneous suspension. Can someone guide me what is the reason for such phenomenon. Partial cell lysis. "Slime" = chromosomal DNA. DK From: d...@no.email.thankstospam.net<mailto:d...@no.email.thankstospam.net> (DK) Subject: Re: Gel Solubilization Buffer Composition Date: 9 November 2017 19:25:54 GMT-5 To: methods@net.bio.net<mailto:methods@net.bio.net> In article <3a598d49-8251-49e6-a147-99e9bb3e5...@googlegroups.com<mailto:3a598d49-8251-49e6-a147-99e9bb3e5...@googlegroups.com>>, angusrbis...@gmail.com<mailto:angusrbis...@gmail.com> wrote: On Saturday, 9 January 2010 18:42:04 UTC, wattne wrote: On 9 Jan., 16:40, Jagadish Katam <jagadish...@hotmail.com<http://hotmail.com>> wrote: Hi, I am working on purifying the DNA by gel extraction procedure and are using the commercially available kits for this purpose. A yellow color gel solubilization buffer is used to dissolve the sliced gel. Here i would know the composition of this gel solubilization buffer. Any suggestions will be greatly appreciated. Thanks and Best Regards, Jag Hi Jag! Sounds like the buffer from QIAGEN, right? Why aren't you more precise?? How should anyone help you if we don't even know what kind of stuff you are using??? You can bet that the buffer composition is secret because otherwise QIAGEN couldn't sell their kit anymore; what do you think? Try a Google, and you will either find out or not. Why should we know the exact formulation???? Thanks and Best Regards, watnne Hi Watnne I found all the other replies to Jag's question helpful and constructive. Yours felt fairly malicious. Please refrain from comments like this in the future, its a waste of time and damages the community feeling of places like this. I don't know about thje feelings but here is the composition: 5.5 M guanidine thiocyanate, 20 mM Tris-HCl, pH 6.6 DK _______________________________________________ Methods mailing list Methods@net.bio.net<mailto:Methods@net.bio.net> http://www.bio.net/biomail/listinfo/methods --------------------------------------------- Barbara MacGregor University of North Carolina Department of Marine Sciences 3117C Venable Hall (a/k/a Murray) CB#3300 Chapel Hill NC 27599 Email bmacg...@unc.edu<mailto:bmacg...@unc.edu> Cell +1 (919) 260-6038 Fax +1 (919) 962-1254 Lab 3110 Venable Hall
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