Production of Electro-Competent Cells

Needed supplies for XL-1 Blue cells

LB/Tet plate (12.5 ug/ml)
LB/Tet solution (12.5 ug/ml) : 10 ml and 1 liter
(autoclave and then add Tet to cooled LB agar or LB broth)
Sterile chilled water (2 l)
Sterile ice cold 10% glycerol (110 ml)
Liquid nitrogen

Note: XL-1 Blue cells are Tet resistant. For other cells such as DH10B, use straight LB as the cells are not antibiotic resistant.

Day 1. Streak an LB/Tet plate (12.5µg/ml) with XL-1 Blue cells

Incubate at 37C overnight

Day 2. Innoculate 5-10 ml of LB/Tet (12.5µg/ml) with 1 colony from overnight plate.

Can also innoculate 100 ml
Incubate at 37C/shaking overnight

Day 3 (the long one)

1. Cell growth

Remove 1 ml of LB for use as spec. blank and put in reference cuvette.
Innoculate 1 liter of LB/Tet solution with 5-10ml of overnight culture. Incubate at 37C/shaking in a 2L flask
Periodically measure growth with a spectrometer until the OD(optical density or absorption at 600nm) reaches 0.5-0.7.
If prewarm LB to 37 C prior to innoculation, this takes about 2.5 hours
Place flask on ice

2. Wash cells

Prepare 250 ml centrifuge bottles (nice graduated ones work well).

Wash and rinse thoroughly 6 x 250ml centrifuge bottles. Place bottles on ice to chill

a. Remove LB

Divide 1L culture into the 6 cold bottles (162 ml each). Spin @ 5,000g, 4C for 20 min
Pour off LB, use 1ml pipet to get all drops of LB. Drain by inverting bottles on paper towels

b. Wash 1 (1000ml total)

Add 170 ml sterile chilled water to each of 6 bottles. Can add 5 ml first to resuspend cells then add more to equalize. Spin @ 5,000g, 4C for 15 min
Pour off water and drain on paper towels. Pellets may be loose. If so drain briefly rather than inverting

c. Wash 2 (350 ml total) and combine to 2 bottles

Add 5 ml of sterile chilled water to each of 6 bottles to resuspend cells
Add 80 ml to 2nd and 3rd bottles and add this to 1st. Bring up to 175 ml
Repeat for other 3 bottles to make 2nd set
Spin @ 5,000g, 4C for 15 min
Pour off and drain

e. Wash 3 (350 ml total)

Add 10 ml water to resuspend cells. Bring up to 175ml sterile chilled water to each of 2 bottles
Spin @ 5,000g, 4C for 15min
Pour off and drain
This procedure can have one more wash step by repeating wash 1. However, I typically skipped this additional step and have had no arcing problems.

3. Resuspend cells

a. Wash in 10% glycerol (100 ml total)

Add 50 ml sterile 10% glycerol to each bottle. Swirl to dissolve pellet
Spin @5000g, 4C for 10 min

b. Resuspend in 10% glycerol (4 ml)

Should estimate pellet volume and add eqaul volume of ice-cold 10% glycerol
Add 2 ml sterile 10% glycerol

c. Aliquot

Pipet 50ul aliquots into cold 1.5ml tubes
Quick freeze by dropping into liquid nitrogen
Can do in Kocherlab dewar or black ice bucket
Remove by hand (latex gloves help some!) and put in -80C as fast as possible

d. Store at -80C.

Based on Short protocols in Molecular Biology section 1.8 (p 1-22). Sambrook suggests doing all rinse steps with 10% glycerol. I had some problems with arcing after using this solution so switched to rinsing with cold water.

4/00 KC