Editing Competent-Cells (section)

== Inoue Method ==

'''LB (Luria-Bertani) Medium'''<br/>Per Liter:To 950 ml of deionized H2O , add Tryptone 10 g, Yeast Extract 5 g, NaCl 10 g. Shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (~0.2 ml). Adjust the volume of the solution to 1 liter with deionized H2O . Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle.



'''SOB Medium'''<br/>Per Liter:To 950 ml of deionized H2O , add: Tryptone 20 g Yeast Extract 5 g NaCl 0.5 g Shake until the solutes have dissolved. Add 10 ml of a 250 mM solution of KCl. (This solution is made by dissolving 1.86 g of KCl in 100 ml of deionized H2O .) Adust the pH of the medium to 7.0 with 5 N NaOH (~0.2 ml). Adjust the volume of the solution to 1 liter with deionized H2O . Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. Just before use, add 5 ml of a sterile solution of 2 M MgCl2. (This solution is made by dissolving 19 g of MgCl2 in 90 ml of deionized H2O . Adjust the volume of the solution to 100 ml with deionized H2O and sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle.)

<br/>'''SOB agar plates '''containing 20 mM MgSO4 and the appropriate antibiotic

<br/>'''Standard SOB''' contains 10 mM MgSO4.

<br/>'''SOB medium''', for growth of culture to be transformed<br/>Prepare three 1-liter flasks of 250 ml each and equilibrate the medium to 18-20ºC before inoculation.

<br/>'''SOC medium'''<br/>Approximately 1 ml of this medium is needed for each transformation reaction. SOC medium is identical to SOB medium except it contains 20 mM glucose. After the SOB medium has been autoclaved, allow it to cool to 60ºC or less. Add 20 ml of a sterile 1 M solution of glucose. (This solution is made by dissolving 18 g of glucose in 90 ml of deionized H2O . After the sugar has dissolved, adjust the volume of the solution to 100 ml with deionized H2O and sterilized by passing it through a 0.22-mm filter.)



=== '''METHOD''' ===

IMPORTANT All steps in this protocol should be carried out aseptically.<br/><br/>'''Preparation of Cells'''

<br/>1. Prepare Inoue transformation buffer (chilled to 0ºC before use). Organic contaminants in the H2O used to prepare transformation buffers can reduce the efficiency of transformation of competent bacteria. H2O obtained directly from a well-serviced Milli-Q filtration system (Millipore) usually gives good results. If problems should arise, treat the deionized H2O with activated charcoal before use.

a. Prepare 0.5 M PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid]) by dissolving 15.1 g of PIPES in 80 ml of pure H2O (Milli-Q, or equivalent). Adjust the pH of the solution to 6.7 with 5 M KOH, and then add pure H2O to bring the final volume to 100 ml. Sterilize the solution by filtration through a disposable prerinsed Nalgene filter (0.45-mm pore size). Divide into aliquots and store frozen at -20ºC.

b. Prepare Inoue transformation buffer by dissolving all of the solutes listed below in 800 ml of pure H2O and then add 20 ml of 0.5 M PIPES (pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter with pure H2O .



c. Sterilize Inoue transformation buffer by filtration through a prerinsed 0.45-mm Nalgene filter. Divide into aliquots and store at -20ºC.

2. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for <span style="color:#FF0000;">16-20 hours at 37ºC</span>. Transfer the colony into 25 ml of LB broth or SOB medium in a 250-ml flask. Incubate the culture for&nbsp;<span style="color:#FF0000;">6-8 hours at 37ºC with vigorous shaking (250-300 rpm)</span>.

3. At about 6 o'clock in the evening, use this starter culture to inoculate three 1-liter flasks, each containing <span style="color:#FF0000;">250 ml of SOB</span>. The first flask receives <span style="color:#FF0000;">10 ml</span> of starter culture, the second receives <span style="color:#FF0000;">4 ml</span>, and the third receives <span style="color:#FF0000;">2 ml</span>. Incubate all three flasks<span style="color:#FF0000;">overnight </span>at <span style="color:#FF0000;">18-22ºC</span> with moderate shaking.

4. The following morning, read the OD600 of all three cultures. Continue to monitor the OD every 45 minutes.

5. When the OD600 of one of the cultures reaches&nbsp;<span style="color:#FF0000;">'''0.55'''</span>, transfer the culture vessel to an ice-water bath for 10 minutes. Discard the two other cultures.

The ambient temperature of most laboratories rises during the day and falls during the night. The number of degrees and the timing of the drop from peak to trough varies depending on the time of year, the number of people working in the laboratory at night, and so on.

Because of this variability, it is difficult to predict the rate at which cultures will grow on any given night. Using three different inocula increases the chances that one of the cultures will be at the correct density after an overnight incubation

6. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4ºC.

7. Pour off the medium and store the open centrifuge bottle on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to walls of the centrifuge bottle or trapped in its neck.

8. Resuspend the cells gently in 80 ml of ice-cold <span style="color:#FF0000;">Inoue transformation buffer</span>.The cells are best suspended by swirling rather than pipetting or vortexing.

9. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4ºC.

10. Pour off the medium and store the open centrifuge tube on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to the walls of the centrifuge tube or trapped in its neck.

'''Freezing of Competent Cells'''

11. Resuspend the cells gently in&nbsp;<span style="color:#FF0000;">20 ml of ice-cold Inoue transformation buffer</span>.

<br/>12. Add <span style="color:#FF0000;">1.5 ml of DMSO</span>. alternatively, add βmercaptoethanol&nbsp; to 20 mM final concentration (add 8 µL&nbsp; of 100%&nbsp;<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">βmercaptoethanol&nbsp;</span>&nbsp;to 20 ml).&nbsp;&nbsp;Mix the bacterial suspension by swirling and then '''<span style="color:#FF0000;">store it in ice for 10 minutes</span>'''.

for competent cells without &nbsp;βmercaptoethanol, add <span style="color:#FF0000;"><span style="background-color:#FFFF00;">2 µl of 0.5 M&nbsp;</span></span><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"></span><span style="color: rgb(255, 0, 0); background-color: rgb(255, 255, 0);">βmercaptoethanol&nbsp;</span><span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;"></span><span style="color:#FF0000;"><span style="background-color:#FFFF00;">to 50&nbsp;&nbsp;&nbsp;µL</span></span> competent cells&nbsp; (20 mΜ final)&nbsp; prior to transformation.

for competent cells without&nbsp; DMSO & β mercaptoethanol, add <span style="color:#FF0000;"><span style="background-color:#FFFF00;">8 µl of 0.5 M βmercaptoethanol and 15 µl DMSO&nbsp; to 200&nbsp; &nbsp;µL&nbsp;</span></span>competent cells&nbsp; (20 mΜ final)&nbsp; prior to transformation.

<br/>13. Working quickly, dispense aliquots of the suspensions into chilled, sterile microfuge tubes. Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of liquid nitrogen. Store the tubes at -70ºC until needed.Freezing in liquid nitrogen enhances transformation efficiency by ~5-fold. For most cloning purposes, 50-ml aliquots of the competent-cell suspension will be more than adequate. However, when large numbers of transformed colonies are required (e.g., when constructing cDNA libraries), larger aliquots may be necessary.

<br/>14. When needed, remove a tube of competent cells from the -70ºC freezer. Thaw the cells by holding the tube in the palm of the hand. Just as the cells thaw, transfer the tube to an ice bath. Store the cells on ice for 10 minutes.

<br/>15. Use a chilled, sterile pipette tip to transfer the competent cells to chilled, sterile 17 x 100-mm polypropylene tubes. Store the cells on ice. Glass tubes should not be used since they lower the efficiency of transformation by ~10-fold

For competent cells without DMSO, add&nbsp;<span style="color:#FF0000;">'''7.5 ul DMSO to 100 ul competent cells after thawing on ice&nbsp;'''</span>

<br/>'''Transformation'''<br/>Include all of the appropriate positive and negative controls

16. Add the transforming DNA (up to 25 ng per 50 ml of competent cells) in a volume not exceeding 5% of that of the competent cells. Swirl the tubes gently several times to mix their contents. Set up at least two control tubes for each transformation experiment, including a tube of competent bacteria that receives a known amount of a standard preparation of superhelical plasmid DNA and a tube of cells that receives no plasmid DNA at all. Store the tubes on ice for 30 minutes.

17. Transfer the tubes to a rack placed in a preheated 42ºC circulating water bath. Store the tubes in the rack for exactly 90 seconds. Do not shake the tubes.Heat shock is a crucial step. It is very important that the cells be raised to exactly the right temperature at the correct rate. The incubation times and temperatures given here have been worked out using Falcon 2059 tubes. Other types of tubes will not necessarily yield equivalent results.

18. Rapidly transfer the tubes to an ice bath. Allow the cells to cool for 1-2 minutes.

19. Add 800 ml of SOC medium to each tube. Warm the cultures to 37ºC in a water bath, and then transfer the tubes to a shaking incubator set at 37ºC. Incubate the cultures for 45 minutes to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.To maximize the efficiency of transformation, gently agitate (<225 cycles/minute) the cells during the recovery period.

20. Transfer the appropriate volume (up to 200 ml per 90-mm plate) of transformed competent cells onto agar SOB medium containing 20 mM MgSO4 and the appropriate antibiotic.When selecting for resistance to tetracycline, the entire transformation mixture may be spread on a single plate (or plated in top agar). In this case, collect the bacteria by centrifuging for 20 seconds at room temperature in a microfuge, and then gently resuspend the cell pellet in 100 ml of SOC medium by tapping the sides of the tube.IMPORTANT Sterilize a bent glass rod by dipping it into ethanol and then in the flame of a Bunsen burner. When the rod has cooled to room temperature, spread the transformed cells gently over the surface of the agar plate. When selecting for resistance to ampicillin, transformed cells should be plated at low density (<104 colonies per 90-mm plate), and the plates should not be incubated for more than 20 hours at 37ºC. The enzyme b-lactamase is secreted into the medium from ampicillin-resistant transformants and can rapidly inactivate the antibiotic in regions surrounding the colonies. Thus, plating cells at high density or incubating them for long periods of time results in the appearance of ampicillin-sensitive satellite colonies. This problem is ameliorated, but not completely eliminated, by using carbenicillin rather than ampicillin in selective media and increasing the concentration of antibiotic from 60 mg/ml to 100 mg/ml. The number of ampicillin-resistant colonies does not increase in linear proportion to the number of cells applied to the plate, perhaps because of growth-inhibiting substances released from the cells killed by the antibiotic

21. Store the plates at room temperature until the liquid has been absorbed.

22. Invert the plates and incubate them at 37ºC. Transformed colonies should appear in 12-16 hours.

=== Prepare Inoue transformation buffer (chilled to 0°C before use). ===
<div>a. Prepare 0.5 M PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid]) by dissolving&nbsp;15.1 g of PIPES in 80 ml of pure H2O (Milli-Q or its equivalent). Adjust the pH of the solution&nbsp;to 6.7 with 5 M KOH and then add pure H2O to bring the final volume to 100 ml.&nbsp;</div><div><br/></div><div>Sterilize the solution by filtration through a disposable prerinsed Nalgene filter (0.45-μm pore size). Divide into aliquots and store frozen at –20°C.</div><div><br/></div><div>b. Prepare Inoue transformation buffer by dissolving all of the solutes listed below in 800 ml&nbsp;of pure H2O and then add 20 ml of 0.5 M PIPES (pH 6.7). Adjust the volume of the Inoue</div><div>transformation buffer to 1 liter with pure H2O.&nbsp;<br/></div><div>
{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Reagent<br/>
| Amount per Liter<br/>
| Final Concentration<br/>
|-
| MnCl2 • 4H2O<br/>
| 10.88 g<br/>
| 55 mM<br/>
|-
| CaCl2 • 2H2O<br/>
| 2.20 g<br/>
| 15 mM<br/>
|-
| KCl<br/>
| 18.65 g<br/>
| 250 mM<br/>
|-
| PIPES (0.5 M, pH 6.7)<br/>
| 20 ml<br/>
| 10 mM<br/>
|-
| H2O to 1 liter<br/>
| <br/>
| <br/>
|}

c. Sterilize Inoue transformation buffer by filtration through a prerinsed 0.45-μm Nalgene filter. Divide into aliquots and store at –20°C.
</div><div>QC Transform<br/></div><div>0.1 pg pUC19: 100 colonies&nbsp;:&nbsp;10<sup>9</sup> cfu/µg<br/></div><div>1 pg pUC19: 100 colonies&nbsp;:&nbsp;10<sup>8 </sup>cfu/µg<br/></div><div>10 pg pUC19: 100 colonies&nbsp;: 10<sup>7</sup> cfu/µg<br/></div>
REFERENCES<br/><br/>Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580.<br/>Hanahan, D. 1985. Techniques for transformation of E. coli. In DNA cloning: A Practical Approach (ed. D.M. Glover), vol. 1 pp. 109-135. IRL Press, Oxford, United Kingdom.<br/>Inoue H., Nojima H., and Okayama H. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene 96: 23-28.&nbsp;NEB