Preparation of XL1 Blue Competent cells
(will work well for BL21, etc as well)
1. The most important thing to remember is to keep the cells as cold as possible at all times! This will insure that the cells are as competent as possible which is very important for quickchange!!
2. Streak out XL1 blue freezer stock on to a LB plate containing 12mg/mL tetracycline.
3. Grow overnight at 37 C.
4. Pick a single colony and inoculate a 5 mL overnight culture using LB/tet.
5. The following day dilute 1 mL of the overnight culture into 100 mL of fresh warm LB, grow in the 37 C shaker to and OD of ~.45.
6. Prepare an ice bath and a dry ice bath.
7. When the culture reaches OD600 of ~.45, chill the culture on ice for 15 minutes.
8. Spin the cells down in chilled falcon tubes in the 4 C centrifuge at a speed of 3500 RPM for about 10 minutes.
9. Pour off the supernatant and re-suspend the cells in 30 mLs cold Tfb1 per 100 mLs.
10. Allow the cultures to chill on the ice bath for 15 minutes.
11. Spin the cells down in the 4 C centrifuge for 15 minutes, at a speed of about 3000 RPM.
12. Pour off the supernatant and resuspend the cells the cells in 5 mL of cold Tfb2 per 100 mL of culture.
13. Chill the cells on ice for 10 minutes.
14. Aliquot 200 uL of cells per sterile eppendorf tube. Crack freeze the cells on the dry ice bath.
Tfb1 solution:
30 mM KoAc ~1.465 grams
100 mM RbCl2 ~6.045 grams
10 mM CaCl2 ~0.735 grams
50 mM MnCl2 ~4.945 grams
15 % glycerol ~75 mL 100% stock
start in ~400 mLs dH2O
pH to 5.8 (~10mL 0.1 M acetic acid)
0.1 M acetic acid = 570 uL glacial acetic acid in 100 mL dH2O
Bring to a final volume of 500 mL and filter sterilize
Tfb2 solution:
10 mM MOPS à 0.210 grams
75 mM CaCl2 à 1.100 grams
10 mM RbCl2 à 0.121 grams
15% glycerol à 15 mL of 100% stock
start in ~75 mLs dH2O
pH to 6.5 - 6.8 with 1N NaOH or 1N KOH
Bring to a final volume of 100 mL.
Filter sterilize.