I know pGEX plasmids are best expressed in BL21 cells for protien expression, and the culture is induced with IPTG, but when I"m initially cloning the plasmid, does it make a difference to transform the ligated product in E.coli such as DH10B or I have these Stellar Cells on hand, to go ahead with colony selection, mini preps, sequencing, etc.
Once the plasmid is confirmed and stock stored- BL21 cells would be used for protien expression/purification?
Thanks for the input!
Hi Katie,
I have no experience with these particular strains, but I think you can use (for cloning and plasmid preparation) almost any strain meeting the following criteria: i) Amp-sensitive (since PGEX-6p-1 is AmpR) ii) not carrying other plasmids (you do not want to deal with mixture of plasmids) iii) having endA1 mutation. As far as I see both your strains meet them :)
Hey Katie
It does not matter what strain of E.coli cells you use for cloning purpose (for transforming your ligation product) as long as these cells are antibiotic sensitive for the antibiotic that you will use as a selection marker for your putative ligated clones. Once confirmed by digestion and sequencing you can purify the plasmid again and transform them into a expression strain BL-21 cells used for expression and purification. Hope this information helps .. Good luck
yes thank you Jagpreet Nanda and Stanislav Kozlovsky for the confirmation!
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