1. Preparation of electrocompetent DH5a cells: autoclave 4 baffled 1 liter flasks containing 500 mL LB. Remove a 1 mL aliquot for OD blanks. Inoculate 3 mL of media with a few μL of frozen DH5a cells obtained by scraping the edge of the frozen cell aliquot with a sterile pipette tip. Grow the cells at 37°C for 12-16 hours at 250 rpm. Inoculate each 500 mL of LB with 500 μL of overnight culture. Remove 300 μL from each flask for an initial OD600determination. Incubate the flasks at 37°C while shaking vigorously at 250-300 rpm to an OD600 of 0.6-0.8 (approximately 1010 cells/mL). Note that Sambrook and Russell[12]recommend an OD600 of 0.35-0.4 (approximately 108 cells/mL.) Transfer the flasks to ice when the cells reach the correct optical density and hold for 15 min. Using sterile technique, transfer 330 mL of the cells to 6 ice cold sterile 500 mL centrifuge bottles. Centrifuge at 4000 x g for 15 min and discard the media. Keep the cells on ice at all times from now on.
2. Wash 1: Gently resuspend each bottle in 1 media volume filter sterilized ice-cold water. Pellet at 4000 x g at 4°C for 15 minutes.
3. Wash 2: Gently resuspend in 0.5 volumes of filter sterilized ice-cold water. Pellet at 4000 x g at 4°C for 15 minutes.
4. Wash 3: Gently resuspend in 0.5 volumes of filter sterilized ice-cold water. Pellet at 4000 x g at 4°C for 15 minutes.
5. Wash 4: Gently resuspend in 0.02 volumes of filter sterilized ice-cold water. Pellet at 4000 x g at 4°C for 15 minutes.
6. Final resuspension: gently resuspend the pellet in 0.002-0.003 volume filter-sterilized ice-cold 10% glycerol by gently swirling the liquid. This results in DNA with a very low ionic strength and high resistance. The typical transformation efficiency (in my hands) is 1.4 x 1010 cfu/μg of supercoiled plasmid. Aliquots of 250 μL (enough for 6 transformation reactions of 40 μL each) should be quick frozen in a dry ice/ethanol bath in microfuge tubes or cryovials and stored at -80°C until required.
7. Electroporation precautions: Do not touch the output or any part of the chamber while engaging the automatic charge and pulse switch. Do not charge the power supply to full voltage of 2500 V when using small gap cuvettes. Wear safety glasses at all times.
8. Optional: Assuming that the construction’s elements contain a single Pst I site in the polylinker, restrict the inactivated ligation reaction with 20 units of Pst I in ligation buffer at 37°C for 20-30 minutes to eliminate residual phosphorylated vector that was only cut with a single enzyme during prep, allowing recircularization during ligation. The Pst I cut will eliminate vectors that contain a polylinker and dramatically reduce background transformants lacking the correct insert.
9. Label 2 sets of siliconized microfuge tubes and a set of Falcon 2057 polystyrene tubes. Chill the microfuge tubes and the 1 mm gap cuvettes on ice. Chill the electroporation cuvette holder in ice. Assemble pipetters and sterile Pasteur pipettes.
10. You should always include a positive control of supercoiled vector DNA. You should also include ligated and unligated vector only controls.
11. Ligation reactions must be diluted in order to reduce the conductivity of the liquid in the cuvette so that arcing does not occur. Dilute the ligation reaction 1:5 by adding 2 μL of the heat-inactivated ligation reaction to 8 μL of ice cold Type I water to one set of microfuge tubes on ice.
12. Thaw the required number of 250 μL aliquots of electrocompetent cells DH5a cells stored at -80°C. You will need 40 μL of cells per transformation. Thaw the cells by setting the cryovial on top of ice. As soon as the cells are thawed, place the tube upright in the ice.
13. Positive control: thaw control DNA (pUC9 monomer at 10 ng/mL = 0.004 ng/2 μL). Dilute 2:10 in water in a chilled microfuge tube. Store on ice. Prepare the required number of cuvettes and place on ice.
14. Gently aliquot 40 μL of competent cells into ice-cold microfuge tubes. Add 2 μL of the 1:5 diluted DNA to each of the following tubes: 1) vector without ligase measures the background consisting of uncut plasmid, 2) vector plus ligase determines the amount of vector that was not dephosphorylated and is able to religate, 3) vector plus ligase, restricted with Pst I, 4) 5:1 insert:vector ligation, restricted with Pst I, 5) 10:1 insert:vector ligation, restricted with Pst I, 6) a supercoiled plasmid DNA positive control. Store the components on ice briefly if necessary.
15. A 5:1 ratio is sufficient if the vector and insert are cut separately (allowing removal of the polylinker fragment via a 30K cartridge) and the concentrations are accurately determined. A 10:1 ratio is recommended if the polylinker is still present in the ligation reaction. If the insert concentration is in doubt, use both ratios or increase the ratio at the risk of double inserts. Higher ratios lead to double-inserts and should be avoided.
16. Refreeze any surplus cells at -80°C in dry ice/ethanol then transfer to a -80°C freezer. Cells may be refrozen once with a 2-fold loss of transformation efficiency.
17. Settings for electroporators for E. coli: BTX model 7200 electroporator: 0.1 cm gap cuvette (BTX P/N 610): use 40 μL of cells at 1.3-1.5 kV/and a fixed 5-6 msecond pulse length with a field strength of 13.0-15.0 kV/cm. BioRad electroporator: 0.1 cm gap cuvette (BioRad 165-2089): 40 μL of cells at 1.8 kV/200 ohms/25 μF with a pulse length of about 5 mseconds 2.5 kV in a 0.1 cm cuvette results in a field strength of 25.0 kV/cm and may lead to arcing. Gibco BRL Cell-Porator: 0.15 cm gap cuvette: use 20-25 μL of cells and 2.4 kV, 4000 ohms/330 μF with a pulse length of about 5 seconds. 2.4 kV in a 0.15 cm cuvette results in a field strength of 16 kV/cm.
18. Immediately after pulsing, add 9 volumes of room temperature SOC to the cuvette and gently transfer the cells to a Falcon 2057 tube. Seconds count. Incubate at 37°C and 250 rpm for 60 min. Plate out multiple 20, 50 and 100 μL aliquots on LB agar supplemented with 100 μg/mL sodium ampicillin, (optional 200 μg/mL methicillin), 0.4 mg/mL 5-bromo-4-chloro-3-indoyl-B-D-galactoside (X-gal) and 0.2 mg/mL isopropylthiogalactoside (IPTG). Triphenyltetrazolium chloride (TTC) can be used at a significant savings to replace the X-gal/IPTG mix if blue/white selection is not required. Typically, several thousand colonies are generated.
19. Small inserts do not generate a definitive X-gal signal. Those colonies should be picked regardless of their color and re-streaked on X-gal plates in order to eliminate problems arising from co-transformation or isolation of two colonies during the initial colony picking step. Three milliliters of 2xYT medium supplemented with 100 μg/mL sodium ampicillin is inoculated from the restreaked colonies and incubated overnight at 250 rpm at 37°C.
20. A 1.5-mL aliquot of the overnight cell culture is transferred to a microfuge tube for plasmid purification via modified alkaline lysis protocols designed to produce suitable templates for fluorescent sequencing.