_______—Module 2 – Transformation of E. coli HB101
and Selection for Kanamycin Resistance
PURPOSE: To transform E. coli strain HB101 with the
ligation products from 1/29/08.
MATERIALS:
PROCEDURE:
1. Prep:
Set water bath to 37ºC
Set water bath to 42ºC.
Ice bucket w/ice
2. Label the microcentrifuge tubes (1.5ml)
“Ligation 1”, “Ligation 2”, “(+) Control”,
and “(+) Control”. Or simply number and
have the key available in your notebook.
3. With a sterile pipette, add 0.25ml ice cold
0.05M CaCl2 into each of the tubes and
place on ice.
4. With a sterile loop, transfer 1 single, well
isolated colony from plate labeled “HB101”
to the tube “Ligation 1.” Twist the loop
vigorously between your fingers to dislodge
the cells.
5. Vortex the cells to mix and fully suspend the
cells in the CaCl2. Place on ice.
6. With a new loop, repeat the procedure with
“Ligation 2”, except transfer two colonies to
the tube.
7. With new loops for each transfer, repeat the
procedure with both “Control” tubes.
8. Dilute the DNA from the ligation reaction by
mixing 5µl of DNA from tube 1 (Module I)
in 45µl qualified water (A). Vortex or tap
tube with finger. Diluting the DNA helps to
minimize the carryover of excess salts from
the ligation reaction.
9. Add 10µl of the diluted ligation reaction DNA
to the tube labeled “Ligation 1” and 10µl of
diluted ligation reaction DNA to the tube
labeled “Ligation 2.” Vortex or tap tubes
with finger.
10. Add 5µl of supercoiled control DNA (2.5
ng/µl) for transformation (E) directly to the
tube labeled “(+) Control”. Vortex or tap
tube with finger to mix.
11. Place all tubes on ice for 15 minutes.
12. Place tubes in 42ºC water bath for 90 seconds.
13. Immediately, return tubes to ice for 2 minutes.
14. Aseptically pipette 0.25ml of recovery broth
into each tube of cells.
15. Incubate cells for 30 minutes in 37ºC water
bath.
16. Place tubes in a microcentrifuge and spin for 1
minute to pellet the cells.
17. Remove and discard 0.4ml of supernatant from
each tube and resuspend cell pellet in
remaining 0.1ml liquid.
18. Label three plates, “(+) Control”, “Ligation 1”,
and “Ligation 2” and pipette the transformed
cells to the corresponding plates.
19. Using a sterile loop for each plate, spread the
cells evenly and thoroughly over the entire
surface. Turn the plate ¼ turn and
thoroughly spread again. Continue untile the
cells are evenly spread.
20. Incubate plates inverted in 37ºC oven
overnight.
21. Estimate the number of transformants on both
plates. Keep track of the counted colonies by
putting a dot over them on the outside of the
plate with a lab marker.
22. Calculate the transformation efficiencies
(number of transformants/µg DNA).
RESULTS:
Number of transformants=______________
Efficiency of transformation=____________