Week 3

Prepare and test competent E. coli cells

In preparation for screening the ligation products later this semester, we are making competent E. coli cells (strain DH5α). We will test the cells to determine their transformation efficiency (i.e. are they competent enough) and store the rest of the cells at -80 °C until we need them.

Take a look at the strain description for DH5α. Why do you think we have chosen this strain?

Read before coming to the lab

Project overview – Step V – Screening newly constructed plasmids

Protocols

General guidelines for molecular biology labs
Preparation and transformation of competent bacteria

To do today:

  • prepare competent cells (steps 2 – 7 in “Preparation and transformation of competent bacteria”)

NOTE: the instructor has completed step 1 - details will be provided in lab.

  • transform the competent cells with supercoiled plasmid (steps 8 – 10) – this is the positive control the protocol refers to

concentration of control plasmid DNA you were given __ ng/µl
amount of plasmid DNA you will add to 100 µl of competent cells µl (add the max amount allowed)
make a negative control (no DNA is added to the cells but the cells are carried through the protocol just like the test cells)

  • while the cells are incubating on ice, prepare the remaining competent cells for storage – see “Storage of competent cells for later use” in "Preparation and transformation of competent bacteria”

Label the tube lids with your group number only.
After snap freezing we will put the competent cells in a -70 °C freezer.

  • plate transformed cells (use SOB plates containing ampicillin at 100 µg/ml)

Plate three dilutions of the transformed cells – each in triplicate (plate 100 µl of 1x, 0.1x and 0.01x dilutions of the cells) – for a total of nine plates of transformed cells

  • plate 100 µl of the negative control on a SOB plate with ampicillin and on a SOB plate with no selection
  • Diagram how you will make the dilutions
diluting%20cells.jpg

To do tomorrow:

Someone from each group will need to come in the day after the lab to:

  • remove plates from the 37 °C incubator,
  • check them for colony growth,
  • bag the plates, and
  • store them at 4 °C.

Unless otherwise stated, the content of this page is licensed under Creative Commons Attribution-ShareAlike 3.0 License