Reagents, supplies, and
coli competent bacteria
- 1.5 ml Eppendorf tubes
- 42°C water bath
- LB broth without ampicillin (no Amp)
- LB + Amp plates
- 100 mM IPTG
- 2% X-gal
- 37°C shaker rack
After ligation of vector and insert is complete, the ligation
mixture is ready to be transformed into bacteria.
- Add 200 µl E.
coli competent bacteria to all of the plasmid ligation
mixture in a 1.5 ml Eppendorf tube. Place on ice for 30
- Heat shock at 42°C X 1 min.
- Add 1 ml of LB broth (No Amp) to cells. Incubate 1 hr at
- Pre-warm 2 LB + Amp plates per transformation at 37°C.
- Transfer a 200 µl aliquot of transformation mixture to a clean
Eppendorf tube (low dilution).
- Microfuge to pellet the cells (Be careful
since too much centrifugation will destroy the
cells). Quickly turn microfuge ON and OFF
3-4X. Pipette off and discard all but 200 µl of solution
- If blue/white color selection can be used, to the low and high
10 µl 100 mM IPTG
50 µl 2% X-gal
- Transfer mixtures to separate, pre-warmed LB + Amp plates.
Spread with a bent glass rod.
- Incubate 8-12 hr, inverted, at 37°C.
- Pick white colonies and suspend in 2.5 ml LB + Amp broth (1
colony per tube). Incubate on shaker rack at 37°C 8 hr to
- Suspensions are ready for mini-prep assay.