This page describes a generic protocol to transform DNA into e.coli bacteria. The protocol includes controls as well.
A plate with e.coli HB 101 colonies grown on top.
For a list of consumables, etc, is implicitly contained in this page (sorry about that).
Day -1 preparations
Prepare 4 LB agar plates, 20ml of LB each, see the Basic Protocols page for reference:
- 1 LB agar
- 2 LB agar / ampicillin
- 1 LB agar / ampicillin / arabinose
The easiest way to do this is to label plates as above, and then:
- prepare 80ml LB agar
- pour 1 plate (20ml), which is the LB agar plate, with 60ml remaining
- add 60µl of ampicillin stock solution
- pour 2 plates at 20ml each, which are the LB agar / ampicillin plates, with 20ml remaining
- add 500µl of stock arabinose solution
- pour 1 plate, which is the LB agar / ampicillin / arabinose plate
Get two test tubes that fit into your heat block, label one -pGLO, the other +pGLO.
Transfer 250µl CaCl2 into each test tube.
Place test tubes on ice.
Transfer a colony of bacteria with an innoculation loop from the e.coli plate into each of the test tubes, but picking up a colony, and then inserting into the test tube and spinning the loop until the bacteria is transferred into the liquid.
Transfer some plasmids from the plasmid DNA tube using an innoculation loop into the test tube marked +pGLO. don’t do this for the other test tube. put the plasmid back to deep freeze after this step.
Leave the test tubes on ice for 10 minutes. Make sure the tubes are in full contact with the ice.
Make sure you have plates with the following labels and corresponding content:
- -pGLO / LB agar
- -pGLO / LB agar / ampicillin
- +pGLO / LB agar / ampicillin
- +pGLO / LB agar / ampicillin / arabinose
Heat shock: put the tubes from the ice into a heat block set at 42 °C for 50 seconds. Then put back on ice for 2 minutes. Do the change rapidly. Make sure the tubes are in good contact with the heat block and the ice as well.
Remove tubes from ice, put on table.
Add 250µl of liquid LB to each test tube.
Leave tubes for 10 minutes at room temperature.
Shake tubes to mix well.
Transfer 100µl of the tube contents each, so that the content from +pGLO go to the two plates marked +pGLO, and from -pGLO to the plates marked -pGLO.
Spread the transferred liquid on top of the plate with an innoculation loop.
Put the plates into a 37 °C incubator
Day +1 results
Look at the plates, and compare the results with the following:
- -pGLO / LB agar: visible bacteria growth, not fluorescent
- -pGLO / LB agar / ampicillin: no bacteria growth
- +pGLO / LB agar / ampicillin: visible bacteria growth, not fluorescent
- +pGLO / LB agar / ampicillin / arabinose: visible bacteria growth, fluorescent