I ordered a bunch of plasmids, and many of them come as agar stabs, where they’ve taken bacteria with the plasmid and stabbed it into a tube of agar. So it’s like a Petri dish in a tube. My gameplan:
- streak a plate
- isolate a colony
- grow a 5mL overnight culture (LB + appropriate antibiotic)
- make a glycerol stock
- take 500μL of that culture & add to 500μL sterile glycerol; invert to mix & store in -80°C freezer – should be good for years if you avoid freeze-thaws
- miniprep the rest of the overnight culture to isolate the plasmid
- send the plasmid for sequencing
- if the sequence checks out, do whatever the heck you got it for! (subclone into a different vector, transform into expression cells, etc.)
links to more:
- more on cloning cells: blog form: https://bit.ly/dh5alpha ; YouTube: https://youtu.be/MwMTFQira4A
- more on expression cells: https://bit.ly/bl21_rare_codons ; YouTube: https://youtu.be/XB3h3Qfe2OE
- more on molecular cloning: http://bit.ly/molecularcloningguide ; YouTube: https://youtu.be/U0O2T7NBUgg
- more on transformation: http://bit.ly/transformheatshock ; YouTube: https://youtu.be/3C6X2a7xWVw
- more on T7 RNAP: http://bit.ly/t7rnap
- more on mini preps: http://bit.ly/minipreps ; YouTube: https://youtu.be/fKf-g5oNvLY
- more on glycerol solution-making: https://youtu.be/iNm1FYKy1lo
- some tips for pipetting & working with viscous solutions: blog: https://bit.ly/pipetting_problems ; YouTube: https://youtu.be/AUTlgR4V6lI
- random practical lab tips & tricks: https://bit.ly/lab_tricks_page
