Tips:
Ampicillin - most definitely add it to the agar, you want a uniform
concentration of the stuff. Top spreading takes forever. Just add
the amp to your agar mix after it's cooled down to merely "hot" and
not boiling.
Amp is decent at selecting from anywhere around 2ug/mL up to
1000ug/mL, we typically use 50-100ug/mL in the lab. A light "dusting"
per plate by eye is generally enough. 100mg/L will work fine as a
recipe.
Amp is very convenient in that you don't have to wait for the
resistance gene to be expressed before adding it. Since it attacks
cell wall growth and not translation, you can just add it. With
kanamycin, chloramphenicol, etc. you have to wait an hour.
---
Re competent cells: I agree that the TSS competency method is
probably the easiest to get to work in a home lab setting.
electrocompetent preps are even easier but you'd then need to
build/obtain a 2500V exponential wave shocker. (Highly recommend
electrocompetent methods if you want really high competency, i.e. if
you ever want to try a random library screen / selection.)
It is absolutely essential that you use "midlog" exponentially growing
cells for the competency prep. You want the cells rapidly dividing so
that their cell wall hasn't yet fully thickened, as it does once you
get to stationary phase. You'll also probably want to keep the
cultures shaking for the exponential growth phase if you want
highly-competent cells. As soon as you stop their growth keep the
cells rigorously cold at 4deg for the best competency.
-A
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