I'm also old fashioned and use a cloning strain for plasmid preps. I've read that derivatives like Bl21 gold are suitable for both purposes. Though, never tried it by myself.
Hughes, Jonathan <jon.hug...@bot3.bio.uni-giessen.de> schrieb am Fr., 10. Juli 2020, 08:38: > Of course! Using a B strain for cloning is just asking for trouble. > Manno... > > > -- > Jon Hughes > Sent without the use of Apple products. > > ---- Jon Cooper schrieb ---- > > Re: "In our lab we generally use BL21 for plasmid replication." > > I am interested because on the occasions that I prepared plasmids in BL21 > (usually by mistake), they were never any good for cloning or sequencing. I > always had to transform them back into a cloning strain and do another > plasmid prep! Probably my incompetence! > > Jon Cooper > > On 9 Jul 2020 21:44, Rafael Marques <rafael_mmsi...@hotmail.com> wrote: > > Hi Umar, > > > > I must say that it would be better use as an expression system Rosetta DE3 > or Rosetta-gami instead of BL21. In our lab we generally use BL21 for > plasmid replication. > > Also, there are a few things that you could try before trying another > construction. > > > > 1. Lower the temperature during the expression. > 2. Try to use a different range of pH in your buffer. Maybe you could > add a bit of NaCl (150 – 300 mM) and/or glycerol (2 – 10%) > 3. I must say that I have already obtained very different results > using Co or Ni columns for IMAC. You could take a look at this. > > > > Regards > > > > ______________________________________________________ > > > > Rafael Marques da Silva > > Mestrando em Física Biomolecular > > Universidade de São Paulo > > > > Bacharel em Ciências Biológicas > > Universidade Federal de São Carlos > > > > phone: +55 16 99766-0021 > > > > * "A sorte acompanha uma mente bem treinada"* > > *________________________________________________* > > > > *De: *Lau Kelvin <kelvin....@epfl.ch> > *Enviado:*quarta-feira, 8 de julho de 2020 16:22 > *Para: *CCP4BB@JISCMAIL.AC.UK > *Assunto: *Re: [ccp4bb] Looking for suggestions with protein expression > > > > Hello Umar, > > > > I would not pin down your difficulties solely due to an Fe-S proteins. I > have produced some with no fusion partners and they work wonderfully. They > were expressed in an aerobic environment and then reduced in an anaerobic > one before usage in reactions. > > > > 1) On the Fe-S side, there are plasmids you can co-transform to increase > Fe-S production. This plasmid pH151 has the synthetic genes necessary for > Fe-S formation. > > https://www.jbc.org/content/279/33/34721.abstract > > > > 2) On the general protein side, have you hhpred your protein? Different > constructs (not just tags), temperature? Strain? Media? > > > > 3) For these proteins we typically use His Excel (or Protein Ark Ni2+ > Advance) that is resistant to most chelators since more often than not, > they contain other metals and can snatch Ni2+ from normal Ni-NTA resins. > Strep tags also work well. > > > > On Jun 27, 2020, at 9:14 AM, Umar Farook <umarfaroo...@gmail.com> wrote: > > > > Dear All, > > > > Sorry for an offtopic question, your suggestions are highly appreciated. > > > > We have been working on iron sulfur cluster binding protein, which is > usually expressed as a nice soluble protein expressed in BL21 cells but > aggregated in the affinity column itself and unable to recover from it. We > had made n number of truncations and fused to soluble tags such as MBP, but > always ended up in large aggregates. Anyone has experience in working with > iron-sulfur cluster binding protein before, please let us know the critical > steps in purification of such proteins, whether you have completely done > the expression, purification and crystallization in anaerobic conditions? > or else changing the expression system to eukaryotic system such as Baculo > or HEK 293T would help? > > > > Please share your valuable experience, thank you. > > > > > > > > -- > > Best Regards, > > Umar Farook > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
