Hi Javier, For a few years (during an industry job) we regularly cloned directly into BL21 (we made our own high competency cells) and confirmed via expression before plasmid sequencing. Only after sequencing did we transform into a cloning strain for miniprep and “long term storage”.
We didn’t see significant issues with RecA but more so over time we lost the DNA to nuclease activity (endA). However, this took months to see any appreciable loss. If your goal is only plasmid recovery, as Jon stated, your current plan should work perfectly. Be sure to use the “optional wash” in your miniprep kit (e.g., Buffer PB in the Qiagen kit) and transform into Top10 within a month and you shouldn’t have significant issues. Best, Nick Clark Nicholas D. Clark (He/Him) PhD Candidate Malkowski Lab University at Buffalo Department of Structural Biology Jacob's School of Medicine & Biomedical Sciences 955 Main Street, RM 5130 Buffalo, NY 14203 Cell: 716-830-1908 On Sun, Feb 18, 2024 at 7:50 PM Javier Gonzalez <bio...@gmail.com> wrote: > Dear all, > I'm sure this issue comes up very often, but for the first time in our lab > we need to recover a pET-type expression plasmid from a BL21-like E. coli > strain (NEB's T7 Express). > I know a RecA+ strain is not suitable for plasmid production, but the > basic plan is to grow and mini-prep the cells to recover the plasmid and > later transform another E coli strain (Top10) to make frozen stocks and for > plasmid production. > Is this a regular practice or is there any known protocol we should follow? > Any advice will be greatly appreciated. > > Best wishes, > Javier > > -- > Dr. Javier M. González > Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET) > Universidad Nacional de Santiago del Estero (UNSE) > RN9, Km 1125. Villa El Zanjón. (G4206XCP) > Santiago del Estero. Argentina > Tel: +54-(0385)-4238352 > Email <bio...@gmail.com> Twitter > <https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Ftwitter.com%2F_biojmg&data=05%7C02%7Cndclark2%40g-mail.buffalo.edu%7C515cc50bc71b4cd8e51508dc30e4c3aa%7C96464a8af8ed40b199e25f6b50a20250%7C0%7C0%7C638439006426537163%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C0%7C%7C%7C&sdata=4ImdcieRx8lB83I7IjI3pb%2FOMQHZdJv0Vw4D6ib5tTQ%3D&reserved=0> > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://nam12.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=05%7C02%7Cndclark2%40g-mail.buffalo.edu%7C515cc50bc71b4cd8e51508dc30e4c3aa%7C96464a8af8ed40b199e25f6b50a20250%7C0%7C0%7C638439006426537163%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C0%7C%7C%7C&sdata=tpIkSwothQw6TdwtLavHhQhf9NiI3pBx%2B8YuwFrG%2FJM%3D&reserved=0> > ######################################################################## 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/
