A Hasty Method to Prepare Competent E. Coli

The method presented here is a modification of Inoue et al (1990). It is suitable for a hasty people like me, because it takes shorter time (finish in late afternoon of the second day) and when to stop bacterial proliferation is easily estimatable.

PROCEDURES
Outline; grow E. coli up to about 0.4 unit O.D.600 at room temperature and replace the medium with a transformation buffer (TB).

1. Day 1, morning. Autoclave (A/C) and dry following lab wares. When you are hasty, dry them (take the covers) in a clean air chamber under ventilation after A/C.
     One or two 165mm glass tubes with polypropylene cap (or disposable sterlie 15mL Falcon tube)
     Two triangular glass flasks with alminium foil cover (one reserve)
     Ten to twelve 2mL microtubes (use tolerant tubes for storage in liquid nitrogen)
     Yellow and blue tips
Prepare SOB, SOC media and TB also, if you have no stock of them in a refrigerator.

2. Day1, late afternoon. Dispense about 2mL SOB to one of the A/C'ed glass tube and 50mL to the two flasks. Inoculate appropriate E. coli strain in glycerol stock into SOB in the glass tube taking 1-2uL of the stock with a yellow tip. Store SOB in the flasks in a refrigerator until use next day. Keep sterile strictly.

3. Shake overnight to grow E. coli at 37C in a water-bath shaker. It will become enough turbid in the next morning.

4. Day 2, morning. Inoculate about 0.5-2mL of the E. coli culture into one of the SOB in a flask. At this moment the medium becomes slightly turbid already. Shake it at room temperature to grow. The rest of E. coli in SOB can be stored as glycerol stock for later use.

5. Day 2, afternoon. Stop growth at about O.D.600 = 0.4. It takes about 2-8hrs to grow to this turbidity, depending on initial bacterial amount added. Check every 10-20min after it has reached about a half of the intended turbidity. It is OK even if it has overgrown. In such a case, inoculate a few mililitters of the overgrown medium into the reserved SOB in another flask, and grow further to appropriate turbidity. It is better to measure O.D. with a photometer. If you are experienced, however, it may be OK to estimate turbidity by eyes because of a wide O.D. 'strike-zone' for high competencies in this method. A turbidity to give good competency may look like a conc consommae soup, thinner than routine overnight culture.

6. Ice cool the bacteral culture in a flask for 5min-1hr. At this point and later on, keep solution containing E. coli ice cold carefully to make highly efficient competent cells. Handle the solution in a clean air chamber. Transfer the culture to a sterile 50mL Falcon tube. Centrifuge at 1000-3000 xG for 10-5min at 4C.

7. During the waiting time of centrifuge, put the 2mL microtubes on a tubestand and put them in a freezer to cool down. Do not over-cool.

8. Discard the supernatant of the centrifuged culture decanting the tube with the bacterial pellet up. Suck a drip at the rim of the tube with a bluetip. Gently add 15mL of ice-chilled TB to the bacterial pellet. Carefully resuspend the bacterial pellet completely with a bluetip blowing the TB onto the pellet. Do not make bubbles.

9. Centrifuge and discard the supernatant as above. Add 5mL of the TB. Resuspend the pellet.

10. Add 350uL of dimethylsulphoxide (DMSO) and gently mix right away. Mixing DMSO with water makes heat, and keep the bacterial solution in an ice bucket. The bacterial cells become competent.

11. Quickly but gently aliquot the competent cell solution dispencing 500uL each to the cooled 2mL microtubes on the tubestand. Be careful not to warm the cell solution.

12. Store the competent cell in liquid nitrogen or in a deep freezer at -80C. Liquid nitrogen stored competent cell is good for about one year without drop of competency in a practical sense.
 

TRANSFORMATION
Outline: mix competent cell solution with plasmid, keep on ice for 30min, give heat shock, and over-grow for 1hr.

1. For conveniency begin the transformation procedures around 3 p.m. Thaw competent cell slowly on ice. Do not shake and over warm. Put aside the melted cell solution on ice.

2. Dispence to put a drop of plasmid solution or ligation reaction mix on the bottom of a 2mL Eppendorf tube. Keep sterile strictly. Put the tube on ice to chill.

3. Pour 100uL of thawed competent cell solution gently onto the ice chilled plasmid or ligation mix solution. Solutions are naturally mixed. Do not mix vigorously.

4. Keep the tube on ice for 25min.

5. Make warm water at 43-44C boiling weakly or with geyser. It takes about 5min to make water warm (25+5=30min.).

6. Put the tube in the warm water on a tube float for 45-50sec.

7. Chill the tube on ice. Immediately proceed to the next step. A lab manual says to keep it for 1-2min. on ice, but it takes about a minute to prepare for the next step.

8. Add 1mL of SOC and shake it for 40-60min at 37C. A lab manual says to pre-warm it to 37C, but it is similarly efficient to pour SOC just taking it out from a refrigerator.

9. While shaking, spread appropriate amount of X-gal and IPTG (c.a. 20 & 10uL + 20-50uL milli-Q water) on one or two LB Amp+ agar plate according to the combination of host strain and vector plasmid. Allow to dry for a while with the cover half opened in a clean air chamber.

10. Spread 100uL of SOC added and overgrown transformant cell solution onto the agar plate. If ligation reaction efficiency is expected to be low, flush down the rest of the solution at 15000 xG to concentrate the cells about 10 times. Discard the most of the medium. Resuspend the cells in the remnant of medium, and spread it onto another agar plate. Otherwise for the safe, it is advisable to set the rest of the transformant aside in a refrigerator until the next day. If number of transformant colonies were too small, you can spread it onto another agar plate concentrating cells in the same way as above.

11. Wrap around the plate cover with parafilm. Incubate it upside-down overnight at 37C. Keep the plate in a refrigerator if bacterial colonies appear in the next morning.

It takes some time for bacterial transformation. If you are hasty, make the steps 4 and 8 shorter. Chill the transforming cell for five min. on ice, and over-grow it for five min. for example, then the transformation efficiency drops to about 1/10. Otherwise, chill it for 15 min. and shake it for five min., then it gives about a half of the standard efficiency. In these cases, pre-warm the SOC. It may be enough to get a small number of clones with good ligation reaction in these hasty ways.



REFERENCE
Inoue H, Nojima H, Okayama H.1990. High efficiency transformation of Escherichia coli with plasmids. Gene 96:23-28.


NOTE

E. coli Strain
DH5alpha, DH5alphaF'IQ, XL1BlueMRF', etc. Handle glycerol stock of either strain under a strict sterile environment, and isolation of single colony is unnecessary. It can thus gain one overnight for incubation of a plate streaking the bacterial stock onto it.
DH5alpha is good for routine transformation. It does not have lacIq, and plating IPTG is unnecessary. Rapid growth gives a good yield of plasmid.
DH5alphaF'IQ is highly competent and good for trying your luck. It needs IPTG for blue-white selection. It grows rather slowly and plasmid yield is smaller. Repeated transplant may cause defection of F' plasmid. Add 2uL kanamycine solution to the small scaled proliferation on the day 1 to keep F' plasmid.
XL1BlueMRF' lacks restriction function of methylated DNAs and is suitable for cloning of genomic DNAs. It needs IPTG for blue-white selection. Repeated transplant may cause defection of F' plasmid. Add 2uL tetracycline solution to the small scaled proliferation on the day 1 to keep F' plasmid.

Glycerol Stock
A/C 80% glucerol (glycerol:milli-Q=8:2). Dispence 500uL 80% glycerol to an Eppendorf tube. Add 300uL E. coli culture, mix well inverting the tube, and flush down. Store in a deep freezer at -80C. Cells survive through a few freeze-thaw cycles (do not thaw completely). Good for about 10 years storage. Very old cell stock may, however become weaker and show slow growth. If so, spread the stock over a LB plate (without or with antibiotics for F' selection), select a viable colony, and prepare a new stock.

Over-Growth
Put tubes on a rotator, put the rotator in a dry incubator set at 37C, and rotate the tubes. Use a big incubator, or it will become too warm for heat generation from the rotator. If there is no big incubator nor rotator, it is OK to use the water-bath shaker with a pre-warmed 50mL Falcon tube in which you can put 2mL tubes with tissue paper as packing. Submerge the Falcon tube over sideways to shake.

Competency Calculation
When 100 colonies appeared on an agar plate from 100uL transformant solution spread, which is made of 100uL competent cell solution and 1uL of 10pg/uL plasmid solution upon 10 times dilution by SOC, the conpetency would be
105+2+1 = 108cfu/ug plasmid.100uL competent cell solution. (10pg x 105 = 1ug, 100 = 102, 101 times diluted)
 

CHEMICALS AND SOLUTIONS

SOB Medium
For 200mL (mix the following broth and Mg solution to 100:1 in a sterile environment, stable for about a year under refrigeration);
Broth
Bacto-tripton                        4g
Yeast extract                        1g
5M NaCl                              0.4mL
3M KCl                               0.167mL
Water                                200mL
A/C

Mg solution
MgSO4.7H2O                            2.465g
MgCl2.6H2O                            2.033g
Add milli-Q water up to 10mL.
A/C

SOC Medium
Mix SOB medium and the following glucose solution to 100:1 in a sterile environment. Stable for a about year under refrigeration.
Glucose                              3.603g
Add milli-Q water up to 10mL.
A/C

TB
For 200mL;
PIPES                                6g
CaCl2.2H2O                            0.44g
3M KCl                               16.63mL

Add about 170mL milli-Q water.
Adjust pH to 6.7 with KOH solution.
Add;
MnCl2.4H2O                            2.18g
Adjust volume to 200mL with milli-Q water.
Sterilize it through 0.2um filter.
Stable for more than a year under refrigeration. Keep it sterile.

LB Amp+ agar plate
For 20-30 plates;
Bacto-trypton                        3g
Yeast extract                        1.5g
5M NaCl                              5.134mL
Agar                                 4.5g
Water                                300mL
A/C
Use an alminium kettle for convenience.

At this point and later on, handle anything sterile in a clean air chamber until agar plates are covered and wrapped.
Cool down the LB agar medium to 60-70C, add 300uL Amp solution, and mix.
Pour 10-15mL of the medium in a disposable plastic plate, and swirl it gently to spread over the bottom of the plate. Cover and stack it.
Pour, swirl, cover and stack one after another, pulsing with flame to avoid clot.
After agar clots, open the plate cover halfway under ventilation of a clean air chamber to dry agar for 30-60min. Do not over-dry.
After steam inside the cover disappeared, close the cover, stack, wrap, and store upside down in a refrigerator.
Good for one or two months under refrigeration. Plates older than two months can be used with about 3-5uL Amp spread with X-gal and IPTG.

5M NaCl (for 1000mL)
NaCl                                 292.5g
Add milli-Q water up to 1000mL.
A/C
Stable for years under refrigeration.

3M KCl (for 100mL)
KCl                                  22.365g
Add milli-Q water up to 100mL.
A/C
Stable for years under refrigeration.

Amp Solution
Ampicilin sodium salt                500mg
50% ethanol                          10mL
Stable for years in a freezer. Handling sterile is unnecessary.

Kanamicine, Tetracycline
Either kanamicine or tetracycline    500mg
Milli-Q water                        10mL
Sterilize through 0.2um filter. Aliquat and store in a freezer. Stable for years. Avoid repeated freeze-thaw cycles.

X-gal
X-gal                                100mg
Dimethylformamide                    5mL
Stable for about a year in a freezer. Longer sotrage makes the solution somewhat colored, but it still works fine.

IPTG
IPTG                                 119mg
Milli-Q water                        5mL
Aliquot and store in a freezer. Good for years.


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