TITLE: Transforming E. coli (Instructor protocol)
AUTHORS: Brian    Teague

[DESCRIPTION]
This is the instructor protocol for
 

Setup for this lab can be pretty intensive if you're starting from scratch. It requires:
Competent E. coli
LB-agar + kanamycin plates
SOC outgrowth media

Notably, commercial competent E. coli are really expensive. We use the Zymo kit, which brings the cost down to ~50 cents per transformation. This protocol makes about 100 transformations, and the cells are competent enough for subcloning, regular restriction-and-ligation cloning, and Golden Gates.

In an attempt to make this work more widely accessible, I have been extremely verbose in these protocols. If you are familiar with recombinant DNA work, you likely have your own protocols for making chemically competent E.coli, pouring plates, and making SOC -- use those!

[STEPS]
SECTION: Prepare kanamycin stock solution
2. 
Weigh 0.5 g of   into a 15 ml conical centrifuge tube.

SECTION: Pour LB-agar + kanamycin plates
12. Fill a 1 liter screw-cap bottle with approximately 900 mL of deionized water.

SECTION: Pour LB-agar + kanamycin plates
14. Add water to a total volume of 1 L (eyeballing is OK, no need to dirty a graduated cylinder). Cap and shake to mix.

 


SECTION: Pour LB-agar + kanamycin plates
15. Loosen the cap and autoclave at 121 °C for 30 minutes on a liquid cycle.

SECTION: Pour LB-agar + kanamycin plates
16. Swirl to mix well, then cool the bottle to at or below 60 °C. You can do this in a water bath, or by swirling under a running cold water tap.

 

 


SECTION: Pour LB-agar + kanamycin plates
17. Optional: pour several plates without any antibiotic. They'll be useful below!
SECTION: Pour LB-agar + kanamycin plates
13. Add:
5 g   
10 g   
10 g   
20 g   
SECTION: Prepare kanamycin stock solution
3. Add 10 mL deionized water to make a stock solution with a concentration of 50 mg/mL. Vortex to dissolve.

SECTION: Prepare kanamycin stock solution
1. Prepare 9 empty microcentrifuge tubes in a rack.
SECTION: Prepare kanamycin stock solution
4. Mount a   on a  . Pull the plunger out of the back and pour the kanamycin solution in.

SECTION: Prepare kanamycin stock solution
5. Holding the syringe filter over the first microcentrifuge tube, insert the plunger back into the syringe. Squeeze the syringe to filter the kanamycin into the waiting tubes. Put about 1.2 mL into each tube. You don't have to be precise, but make sure there's at least 1 mL in each.

SECTION: Prepare kanamycin stock solution
6. Use immediately or store at -20 °C .

SECTION: Pour LB-agar + kanamycin plates
18. Add 1 mL of 50 mg/mL kanamycin solution and swirl to mix well.

SECTION: Pour LB-agar + kanamycin plates
19. Option A: Pour ~15 ml of molten media into each  , enough to cover the bottom . If you make 1 L of media, you'll use about 2 sleeves (25/sleeve).

SECTION: Pour LB-agar + kanamycin plates
20. Option B: Using a 25 ml pipette and a pipettor, pipette ~15 ml of molten media into each  , enough to cover the bottom . If you make 1 L of media, you'll use about 2 sleeves (25/sleeve).

SECTION: Pour LB-agar + kanamycin plates
21. Leave the plates   on the bench to cool.

SECTION: Pour LB-agar + kanamycin plates
22. Put the petri dishes back in their plastic bags and store inverted at 4 °C . Plates are good for at least 3 months.

SECTION: Making SOB and SOC outgrowth media
7. In a 250 ml bottle, add approximately 200 mL deionized H2O.

SECTION: Making SOB and SOC outgrowth media
8. Add to the bottle:
5 g   
1.25 g   
0.145 g   (or 0.5 mL of a 5 Molarity (M) solution)
0.0475 g   (or 125 µL of a 1 Molarity (M) solution)
2.5 mL 1 Molarity (M) solution of   
2.5 mL 1 Molarity (M) solution of   






SECTION: Making SOB and SOC outgrowth media
9. Add deionized water to a final volume of 250 ml. (You can eyeball it -- no need to dirty a graduated cylinder).
SECTION: Making SOB and SOC outgrowth media
10. Autoclave at 121 °Con a liquid cycle for 30 min. (This media is SOB - "Super Optimal Broth")

SECTION: Making SOB and SOC outgrowth media
11. Using good sterile technique, make 5 mL aliquots into 15 ml conical centrifuge tubes, 1 per 4 people. To each aliquot, add 50 µL40 Mass / % volume  . (These aliquots are SOC - "Super Optimal Broth, Catabolic")
 





SECTION: Making chemically competent E. coli
23. Two days before the prep, strike out the E. coli cloning strain (from a freezer stock) on an LB-agar plate (no antibiotics!). Incubate at 37 °C30 min .

SECTION: Making chemically competent E. coli
24. The afternoon before the prep, pick a colony off of the plate and start an overnight culture in 5 mL    (in a round-bottomed test tube.) Shake   30 min 

SECTION: Instructor Tips &amp; Common Student Errors
46. Instructor Tips
Do not decrease the incubation times, especially for the outgrowth. I tried decreasing the outgrowth time to 30 minutes, once, and every single transformation failed. Kanamycin is not like ampicillin -- it doesn't just inhibit growth, it actually kills the cells. So they need to be expressing the resistance gene before they are challenged with the antibiotic.
I recommend testing the competent cells to make sure they are actually competent before handing them to your students.
Especially the first time, a two-hour lab may not be enough time for the 90 minutes of (total) incubation. I will often instruct students to label and prepare their plates with beads and leave them on the bench for me -- and then, after their incubations are done, I'll plate their cells for them. Timing isn't important here -- I've seen successful transformations after even 3 hours of outgrowth.
Make sure the water bath and incubator are turned on well before lab starts -- they take a while to come to temperature!
If you're using a dry bath instead of a big water bath, fill the dry bath's wells with water to increase the efficiency of heat transfer.
If you don't have an incubating shaker, it's not the end of the world -- a 37 °C incubator is probably fine.

SECTION: Making chemically competent E. coli
25. Transfer 50 ml of SOB (above) to a 250 ml baffled flask.

SECTION: Instructor Tips &amp; Common Student Errors
47. Common Student Errors
Didn't check the SOC and used a contaminated tube.
Added the positive control AND their ligation to the competent E. coli.
SECTION: Making chemically competent E. coli
26. Add 0.246 g  

 

SECTION: Making chemically competent E. coli
27. Add 0.5 mL of the overnight culture.

SECTION: Making chemically competent E. coli
28. Fold a piece of aluminium foil over the mouth of the flask. Shake   until the OD600 of the culture reaches 0.4-0.6. This usually takes about 3 hours.

 


SECTION: Making chemically competent E. coli
29. While the culture is growing:
Chill a swinging-bucket or high-speed centrifuge to 4 °C 
Load 96 microcentrifuge tubes into a 96-position tube rack and place them in the cold room to chill (or on ice). Tent a paper towel over them to keep out contaminating microbes
From the   kit, mix 2.5 ml of 2X Wash Buffer and 2.5 ml Dilution Buffer to prepare 5 ml of Wash Buffer in a conical centrifuge tube. Keep on ice.
From the   kit, mix 2.5 ml of 2X Competent Buffer and 2.5 ml Dilution Buffer to prepare 5 ml of Competent Buffer in a conical centrifuge tube. Keep on ice.
Pre-chill a 50 ml conical centrifuge tube on ice.

SECTION: Making chemically competent E. coli
30. When the culture has reached an OD600 of between 0.4 and 0.6, transfer the baffled flask to an ice bucket and mound up the ice around the flask. Chill for 10 minutes.

 

SECTION: Making chemically competent E. coli
31. Transfer the culture from the 250 ml baffled flask to the 50 ml pre-chilled conical centrifuge tube. Centrifuge in a pre-chilled swinging-bucket centrifuge (or high-speed fixed-angle centrifuge) 2000 x g, 10 min, 4 °C .

 


SECTION: Making chemically competent E. coli
32. In the cold room (if available), decant the media back into the baffled flask, then invert the 50 ml conical onto a paper towel for a minute to let the media drain away.
SECTION: Making chemically competent E. coli
33. Resuspend in 5 ml 1X Mix&Go Wash Buffer by gentle votexing (ie, on a setting of 3-4). Be patient, it will take a few minutes for the cells to resuspend at this speed. Put the resuspended cells back on ice.

 

SECTION: Making chemically competent E. coli
34. Centrifuge in a pre-chilled swinging bucket (or high-speed fixed-angle) centrifuge 2000 x g, 5 min, 4 °C 

SECTION: Making chemically competent E. coli
35. In the cold room, decant the wash buffer, then invert the 50 ml conical onto a paper towel for a minute to let the wash buffer drain away.
SECTION: Making chemically competent E. coli
36. Resuspend the pellet in 5 ml 1X Mix&Go Competent Buffer by gentle votexing (ie, on a setting of 3-4). Be patient, it will take a few minutes for the cells to resuspend at this speed. Put the resuspended cells back on ice.
SECTION: Making chemically competent E. coli
37. Pipette 50 ul aliquots into the prepared microcentrifuge tubes.
 

SECTION: Making chemically competent E. coli
38. Optional but highly recommended - snap-freeze the cells in liquid nitrogen.
SECTION: Making chemically competent E. coli
39. Transfer the tubes to a -80°C freezer, trying to minimize the time between cold room and freezer.
 

SECTION: Prepare the transformation control
40. Two days before: strike out the E. coli harboring the YTK96 plasmid on a LB agar + kanamycin plate
SECTION: Prepare the transformation control
41. The day before: Pick one colony of the YTK96-harboring E. coli into 5 ml LB broth + 50 ug/ml kanamycin.
SECTION: Prepare the transformation control
42. Miniprep the plasmid, using   or comparable.

SECTION: Prepare the transformation control
43. Analyze the concentration and purity of the miniprep using a Nanodrop.
SECTION: Prepare the transformation control
44. Prepare transformation controls by diluting the YTK96 miniprep to a concentration of 1 ng/ul in   or similar.
 

 


SECTION: Miscellaneous preparation
45. Pour the   into several 15 ml conical centrifuge tubes.