Competent Cells

Using Frozen Competent Cells

"Long" Transformation

1)        thaw competent cells on ice until just liquid, use 200 ul per transformation

2)        add DNA up to 20 ul and swirl to mix

3)        incubate on ice 30 minutes

4)        heat shock at 42 C for 90 sec, then place on ice briefly

5)        add 800 ul LB and incubate at 37 C for 45 minutes

6)        plate out under appropriate conditions

5 Minute Transformation

1)        Thaw competent cells on ice until just liquid.  Use 200ul per transformation.

2)        Add DNA to 20ul and swirl to mix.

3)        Incubate on ice 5 minutes.

4)        Plate on pre-warmed LB+antibiotic plates, one plate with about 20ul and another plate with 100ul .

Which competent cells do I use?

DH5a - general purpose.  Use for transforming plasmids.

XL10 Gold – Large or ligated DNA

XL1 Blue – unlimited DNA or sublconing, unmethylated DNA, specific protocols

Preparation

To make plate in step 1, use old competent cell stock.  Streak a small amount of frozen cells onto LB plate.

1)        pick one colony off fresh DH5a (or other competent cell) plate into 2.5 ml LB supplemented with 25 ul 1 M MgSO4(10 mM final conc.) 

2)        shake at 37 C overnight and until use

3)        do a 1:500 dilution by inoculating 100 ml of SOB in 1 L flask with 200 ul of o/n DH5a, record start time

4)        spec starting at 3 hours after start time to an OD550 0.15 to 0.3 or “eye spec” every 20 minutes starting at 3 hours after start time.

5)        collect in two pre-cooled 50 ml orange cap c/f tubes and incubate on ice 15 min

6)        pellet in the hermle swinging bucket rotor at 2500 rpm for 5 min at 4 C with no brake, drain pellet thoroughly

7)        resuspend in RF1 in 1/3 original volume(30 ml) by gently pipetting with DNA tips

8)        pellet in the hermle swinging bucket rotor at 2500 rpm for 5 min at 4 C with no brake, drain pellet thoroughly

9)        resuspend by gently swirling in RF2 in 1/12.5 original volume(8 ml) and incubate on ice 15 min

10)     aliquot into pre-cooled tubes, 20 tubes with 200 ul and 10 tubes with 400 ul

11)     quick freeze in LN2, using a different colored sharpie than the previous batch mark(see detail table this section) 200 ul tubes with one slash and 400 ul tubes with two slashes

12)     store at -70 C

Notes:

Š           once iced keep cells cool and pre-cool all tubes

Š           be gentle, don’t vortex or roughly pipet cells

Š           use forceps to lower tubes into LN2 in case tubes leak

RF1                                                    1 Liter

100mM  RbCl                                   12g

50mM MnCl2 -4H2O                         9.9g

30mM KAc    -                                   30ml of 1M pH7.5

10mM CaCl2 2H2O                          1.5g

15% w/v Glycerol                             150ml

Adjust to pH5.8 w/ 0.2M acetic acid.  Sterilize by filtration.  Store at 4°C.

RF2                                                    1 Liter                         100mL

MOPS (10mM)                                 2.09g                          0.21g

RbCl                                                   1.2g                            0.12g             

CaCl2 - 2H2O                                     11g                             1.1g

15% w/v Glycerol                             150g                           15g