MicroPulser™ Electroporation Apparatus Operating Instructions and
Contents
1. Number Product Description Mini packs 165 2083 MicroPulser Gene Pulser Cuvettes 0 1cm gap 5 sterile 165 2082 MicroPulser Gene Pulser Cuvettes 0 2cm gap 5 sterile 165 2081 MicroPulser Gene Pulser Cuvettes 0 4cm gap 5 sterile Standard Packs 165 2089 165 2086 165 2088 Jumbo Packs 165 2093 165 2092 165 2091 25 MicroPulser Gene Pulser Cuvettes 0 1cm gap 50 sterile MicroPulser Gene Pulser Cuvettes 0 2cm gap 50 sterile MicroPulser Gene Pulser Cuvettes 0 4cm gap 50 sterile MicroPulser Gene Pulser Cuvettes 0 1cm gap 500 sterile MicroPulser Gene Pulser Cuvettes 0 4cm gap 500 sterile MicroPulser Gene Pulser Cuvettes 0 2cm gap 500 sterile Catalog Number Product Description Plasmid purification 732 6100 732 6120 732 6130 Quantum Prep Miniprep Kit 100 preps includes 20 ml cell resuspension buffer 25 ml cell lysis buffer 25 ml neutralization buffer 20 ml Quantum Prep matrix 63 ml wash buffer 100 mini spin filters instructions Quantum Prep Midiprep Kit 20 preps includes 110 ml cell resuspension buffer 110 ml cell lysis buffer 110 ml neutralization buffer 20 ml Quantum Prep matrix 125 ml wash buffer 20 midi spin filters instructions Quantum Prep Maxiprep Kit 10 preps includes 165 ml cell resuspension buffer 250 ml cell lysis buffer 165 ml neutralization buffer 110 ml Quantum Prep matrix 270 ml wash buffer 10 midi spin filters instructions 26 Life Science Group Bi2. ss224 sceitascce Sis aces ciaasttiars Shasscasccaysiascseetessssoten oc eiesns aiaeves sesso ees 20 Solutions and reagents for electroporation ccceceseeseseeseseeseseeseeseseeseseeseseeeeaeees 20 Appendix I References Appendix II Troubleshooting Guide for the MicroPulser sssscccssssssssssessoees Appendix III Product Information Warranty Bio Rad Laboratories warrants the MicroPulser against defects in materials and workmanship for 1 year If any defects occur in the instrument during this warranty period Bio Rad Laboratories will at Bio Rad s option repair or replace the defective parts free of charge The following defects however are specifically excluded l 2 3 4 5 6 Defects caused by improper operation Repair or modification done by anyone other than Bio Rad Laboratories or an authorized agent Use of fittings or other spare parts supplied by anyone other than Bio Rad Laboratories Damage caused by accident or misuse Damage caused by disaster Corrosion due to use of improper solvent or sample For any inquiry or request for repair service contact Bio Rad Laboratories after confirming the model serial number invoice number and purchase order number of your instrument Model Catalog No Date of Delivery Serial No Invoice No Purchase Order No Section 1 Safety Information Read This Information Carefully Before Using The
3. Should an arc occur the sample chamber is effective in containing these small discharges but nonetheless we strongly recommend wearing safety glasses when using the instrument Do not use the MicroPulser with samples suspended in conductive media refer to Section 3 3 for information on sample resistance 1 3 Other Safety Precautions Turn the unit off when not attended Avoid spilling any liquids onto the apparatus Use only a paper towel or a cloth wet with either water or alcohol to clean the outside surfaces of the MicroPulser Use only the Bio Rad cables supplied with the MicroPulser Only use the shocking chamber in the assembled condition Do not attempt to circumvent the protection of the shocking chamber or use it while disassembled Verify the display segments periodically Do not use the MicroPulser if obvious case damage exists that exposes part of the inside of the unit No user serviceable parts are contained within the MicroPulser the case should only be opened by properly trained personnel Warning The MicroPulser generates uses and radiates radio frequency energy If it is not used in accordance with the instructions given in this manual it may cause interference with radio communications The MicroPulser has been tested and found to comply with the limits for Class A computing devices pursuant to Subpart J of Part 15 of FCC Rules which provide reasonable protection against such interference when operated in
4. and if no P is given there is a single pulse 2 Using The Micropulser in Manual Mode A To change the voltage Press the Settings button to illuminate the LED next to Manual The display LED now shows the voltage in kV Pressing the Raise and Lower buttons to the left of the display LED allows selection of the desired voltage in the range from 0 20 kV to 3 00 kV If the instrument was just turned on the display LED shows 0 00 B To truncate the pulse Press both Raise and Lower buttons simultaneously while the Manual LED is illuminated The display LED now shows t and indicates the pulse time that has been selected for the pulse The default setting when the power is turned on is the standard exponential decay pulse or no pulse truncation which is indicated by the two dashes Releasing only the Lower button results in the display LED showing the time of the truncated pulse in milliseconds The display LED initially changes to t1 0 and rises in 0 1 msec increments to t4 0 This permits truncating the exponential decay pulse between 1 4 msec Simultaneously pressing both Raise and Lower buttons then releasing only the Raise button results in lowering the indicated truncation time 10 3 Pulse Function Pressing the Pulse button results in the capacitor charging to the set voltage during this time PLS is shown on the display LED A tone will sound to indicate that the pulse has been deliver
5. MicroPulser The MicroPulser meets the safety requirements of EN61010 and the EMC requirements of EN61326 for Class B including flicker and harmonics 1 1 Electrical Hazards The MicroPulser produces voltages up to 3 000 volts and is capable of passing very high currents When charged to maximum voltage the instrument stores about 50 joules A certain degree of respect is required for energy levels of this order Safety system features prevent operator access to the recessed input jacks and to the recessed electrode contacts inside the sample chamber These mechanical interlocks should never be circumvented There is high voltage present whenever the yellow pulse button is depressed and PLS is shown in the light emitting diode display on the front of the instrument If the capacitor has been partially charged but not fired for example when the charging cycle has been interrupted before the pulse is delivered some charge may remain on the internal capacitor However the user cannot make contact due to the system safety features 1 2 Mechanical Hazards The MicroPulser contains a patented arc protection circuit that dramatically reduces the incidence of arcing in the cuvette when high voltage is delivered into the sample The unit incorporates a circuit which senses the beginning of an arc and diverts current from the sample within 5 usec preventing or greatly reducing mechanical visual and auditory phenomena at the shocking chamber
6. Section 8 Electroporation of Saccharomyces cerevisciae 8 1 Preparation of Electrocompetent Cells See Becker amp Guarantee 1991 and Ausubel et al 1987 for additional information 1 Inoculate 500 ml of YPD in a 2 8 L Fernbach flask with an aliquot from an overnight culture of S cerevisiae The doubling time of S cerevisiae is approximately 2 hrs at 30 C 2 Incubate at 30 C overnight shaking at 250 rpm to a density of 1 x 108 cells ml 3 Chill the cells in an ice water bath for 15 min to stop growth 4 Decant the cells into two sterile 250 ml centrifuge bottles and pellet the cells by centrifugation at 3000 x g for 5 min at 4 C 5 Carefully pour off and discard the supernatant place the centrifuge bottles with the cell pellets on ice 6 Add 50 ml of sterile ice cold water to each of the bottles and vortex to resuspend the cell pellets bring the volume in each of the centrifuge bottles to 250 ml Pellet the cells by cen trifugation at 3000 x g for 5 min at 4 C pour off and discard the supernatant 7 Wash the cells again as in step 6 with a total of 250 ml sterile ice cold water 8 Resuspend the cell pellet in 20 ml of sterile ice cold 1 M sorbitol and transfer to a chilled 30 ml Oakridge tube Pellet the cells by centrifugation at 3000 x g for 5 min at 4 C pour off and discard the supernatant 9 Resuspend the cell pellet in 0 5 ml of sterile ice cold 1 M sorbitol the final cell volume should be 1 3 m
7. Somkuti G A and Steinberg Genetic transformation of Streptococcus thermophilus by electro poration Biochimie 70 579 1988 Summers D K and Withers H L Electrotransfer direct transfer of bacterial plasmid DNA by electroporation Nuc Acids Res 18 2192 1990 Taketo A DNA transfection of Escherichia coli by electroporation Biochim Biophys Acta 949 318 1988 Taketo A RNA transfection of Escherichia coli by electroporation Biochim Biophys Acta 1007 127 1989 Trevors J T Chassy B M Dower W J and Blaschek H P Electrotransformation of bacteria by plasmid DNA in Guide to Electroporation and Electrofusion Chang D C Chassy B M Saunders J A and Sowers A E eds Academic Press Inc 265 San Diego 1992 Willson T A and Gough N M High voltage E coli electro transformation with DNA follow ing ligation Nuc Acids Res 16 11820 1988 Zabarovsky E R and Winberg G High efficiency electroporation of ligated DNA into bacteria Nuc Acids Res 18 5912 1990 Appendix Il Troubleshooting Guide for the Micropulser Operational Problem Possible cause and solution 1 Display does not light Power is not supplied to electronics when unit is turned on Check power cord and wall outlet power source 2 Check that power switch is on Check replace fuse Two 2 A 250 V Type T fuses are located on the back of the instrument immedi ately above the power cord When the
8. a commercial environment Operation of this equipment in a residential area is likely to cause interference In this case the user will be required at their own expense to take whatever measure may be required to correct the interference Section 2 Introduction 2 1 Overview of Electroporation Theory The MicroPulser system is used for the electroporation of bacteria yeast and other microorganisms where a high voltage electrical pulse is applied to a sample suspended in a small volume of high resistance media The system consists of a pulse generator module a shocking chamber and a cuvette with incorporated electrodes Figure 1 The sample is placed between the electrodes in the cuvette The MicroPulser module contains a capacitor which is charged to a high voltage the module then discharges the current in the capacitor into the sample in the cuvette i bahi a i Fig 1 MicroPulser consisting of Pulse Generator Module Shocking Chamber and Cuvette The capacitance discharge circuit of the MicroPulser generates an electrical pulse with an exponential decay waveform Figure 2 When the capacitor is discharged into the sample the voltage across the electrodes rises rapidly to the peak voltage also known as the initial voltage V and declines over time t as follows V V e Equation 1 where T R x C the time constant a convenient expression of the pulse length The resistance of the circuit R is expresse
9. and vortex to resuspend the cell pellets add 1 25 ml of 1M DTT to each bottle mix gently Incubate the cells for 15 min at 30 C Add 200 ml of sterile ice cold 1 M sorbitol to each centrifuge bottle Pellet the cells by centrifugation at 3000 x g for 5 min at 4 C pour off and discard the supernatant Add 50 ml of sterile ice cold 1 M sorbitol to each of the bottles and vortex to resuspend the cell pellets bring the volume in each of the centrifuge bottles to 250 ml with sterile ice cold 1 M sorbitol Pellet the cells by centrifugation at 3000 x g for 5 min at 4 C pour off and discard the supernatant Resuspend each cell pellet in 10 ml of sterile ice cold 1 M sorbitol and pool in a chilled 30 ml Oakridge tube Pellet the cells by centrifugation at 3000 x g for 5 min at 4 C pour off and discard the supernatant Resuspend the cell pellet in 0 5 ml of sterile ice cold 1 M sorbitol the final cell volume should be 1 3 ml and the cell concentration should be 1 x 10 cells ml Keep the cells on ice and use as soon as possible for electroporation 11 2 Electroporation 1 Pipette the DNA samples up to 10 ug to be electroporated into sterile 1 5 ml microfuge tubes Place tubes on ice Add 40 ul of the competent cells to each DNA sample and mix gently Set the MicroPulser to Pic See Section 4 for operating instructions Transfer the DNA ell samples to 0 2 cm electroporation cuvettes that have been chilled in ice and
10. kV and a time constant of 5 msec For electroporation of S cerevisiae conditions reported as being used most often are 0 2 cm cuvettes containing 40 pl of cells at a voltage of 1 5 kV and a time constant of 5 msec For many bacterial species including Salmonella Pseudomonas Helicobacter Borrelia Streptococcus Lactococcus and Enterococcus the conditions for electroporation are identical to those used for E coli For many other bacterial species altering the field strength will often result in higher electrotransformation A similar case is found with other species of yeast The MicroPulser is designed to deliver precisely those pulse parameters needed for the highest transformation efficiency of E coli and S cerevisiae The time constant has been set 5 at 5 milliseconds when working with high resistance samples For these organisms the MicroPulser has pre programmed settings for delivery of the correct voltage when electroporating E coli in either 0 1 or 0 2 cm cuvettes or when electroporating S cerevisiae in either 0 2 or 0 4 cm cuvettes 3 1 Cell Growth For most bacterial species the highest transformation efficiencies are obtained when cells are harvested in early to mid log growth For E coli as the cells reach stationary phase the transformation efficiency will decline precipitously Dower 1990 In contrast most yeast species are generally harvested in mid to late log growth For S cerevisiae the transformation e
11. tap the suspension to the bottom of the tube Place the cuvette in the chamber slide Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber Pulse once Remove the cuvette from the chamber and immediately add 1 0 ml of ice cold 1 0 M sorbitol to the cuvette when selecting for complementation of an auxotrophic mutant or 1 0 ml of ice cold YPD sorbitol when selecting for antibiotic resistance Gently transfer the diluted cells to a sterile tube Check and record the pulse parameters The time constant should be close to 5 milliseconds The field strength can be calculated as actual volts kV cuvette gap cm When selecting for complementation of an auxotrophic mutant the cells may be plated immediately onto minimal agar plates containing 1 M sorbitol but lacking the appropriate nutrient When selecting for antibiotic resistance incubate the cells at 30 C for 1 2 hrs without shaking plate aliquots of the electroporated cells on YPD agar plates containing 1 M sorbitol with the appropriate antibiotic Incubate the plates for 72 96 hrs at 30 C 11 3 Solutions and Reagents for Electroporation l 2 YPD HEPES 100 ml YPD media 20 ml 1 M HEPES pH 8 0 1M DTT 1 55 g dithiothreitol dissolve in 8 ml water Bring the volume to 10 ml with water Filter sterilize YPD sorbitol 10 g yeast extract 20 g peptone 182 2 g sorbitol dissolve in 700 ml water bring volume to 900 ml with
12. the display LED between the Actual voltage and the Time constant 4 3 Electroporation Using The MicroPulser 1 Place the cell suspension in an electroporation cuvette and tap the liquid to the bottom of the cuvette Up to 0 4 ml 400 ul of solution may be placed in the 0 2 cm cuvette and up to 80 ul may be placed in the 0 1 cm cuvette Note that temperature may have a significant influence on transformation frequency Electroporation of some organisms including E coli and S cerevisiae is more efficient in chilled cuvettes 2 Insert the cuvette into the slide of the shocking chamber Push the slide into the chamber until the cuvette makes firm contact with the chamber electrodes 3 To charge the capacitor and deliver a pulse press the yellow Pulse button the display LED will show PLS until a tone sounds indicating that the pulse has been given The display LED will then show the program the time constant or the actual volts delivered depending on the LED selected 4 Withdraw the slide from the chamber remove the cuvette and process the sample 5 The time constant and the actual voltage delivered to the sample are shown on the display LED by pressing the Measurements button When the LED next to Actual kV is illuminated the voltage is displayed in kilovolts The time constant can be displayed by pressing the Measurements button again The LED next to Time ms will be illuminated the time constant is displayed in mill
13. water Autoclave Add 100 ml sterile 20 glucose 20 Appendix I References 1 Allen S P and Blaschek H P Factors involved in the electroporation induced transformation of Clostridium perfringens Appl Environ Microbiol 54 2322 1990 2 Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K eds Current Protocols in Molecular Biology John Wiley amp Sons NY 1987 3 Becker D M and Guarente L High efficiency transformation of yeast by electroporation Methods Enzymol 194 182 1991 4 B ttger E C High efficiency generation of plasmid cDNA libraries using electro transformation BioTechniques 6 878 1988 5 Chang D C Chassy B M Saunders J A and Sowers A E eds Guide to Electroporation and Electrofusion Academic Press Inc San Diego 1992 6 Cregg J M and Russell K A Transformation in Methods in Molecular Biology 103 Higgins D R and Cregg J M eds Humana Press Totowa NJ 27 1998 7 Cruz Rodz A L and Gilmore M S High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation Mol Gen Genet 224 152 1990 8 Dennis J J and Sokol P A Electrotransformation of Pseudomonas in Methods in Molecular Biology 47 Nickoloff J A ed Humana Press Totowa NJ 125 1995 9 Dower W J Electroporation of bacteria a general approach to genetic transformation in Genetic Engine
14. 0 min Plate aliquots of the electroporated cells on minimal agar plates containing 1 2 M sorbitol Incubate plates for 60 96 hrs at 30 C 9 3 Solutions and Reagents for Electroporation l 2 YCD media 10 g yeast extract 2 g casamino acids dissolve in 900 ml water Autoclave Add 100 ml 20 glucose 1 2 M sorbitol 218 6 g sorbitol dissolve in 700 ml water Add water to 1 0 L Section 10 Electroporation of D discoideum 10 1 Preparation of Electrocompetent Cells See Howard et al 1988 and Knecht amp Pang 1995 for additional information Inoculate D discoideum cells at a concentration of 5 7 x 10 cells ml into 40 ml of HL5 media ina 500 ml flask The cells may either be scraped from a plate or transferred from liquid media The doubling time of D discoideum is approximately 12 hrs at 21 C Incubate the culture at 21 C for about 24 hrs shaking at 125 rpm About 16 20 hrs prior to preparing the competent cells dilute the cells to 7 x 10 cells ml with HL5 media Incubate at 21 C overnight shaking at 125 rpm Transfer 100 ml of the cells into two sterile disposable 50 ml centrifuge tubes and incu bate on ice for 15 min to stop growth Pellet the cells by centrifugation at 400 x g for 5 7 min at room temperature Carefully pour off and discard the supernatant place the centrifuge bottles with the cell pellets on ice Pool the cell pellets and resuspend in 50 ml of sterile ice cold E buffer Pellet t
15. A sample to be electroporated add 1 ml of YM broth to a 17 x 100 tube at room temperature and place a 0 1 cm electroporation cuvette on ice 3 Thaw the electrocompetent A tumefaciens cells on ice For each DNA sample to be electroporated add 20 ul of electrocompetent cells to each DNA sample gently tap the tubes to mix 4 Set the MicroPulser to Agr See Section 4 for operating instructions 5 Transfer the DNA cell samples to the electroporation cuvettes and tap the suspension to the bottom of the tube Place the cuvette in the chamber slide Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber Pulse once 6 Remove the cuvette from the chamber and immediately use the YM broth in the 17 x 100 mm tube to transfer the cells from the cuvette to the tube 7 Check and record the pulse parameters The time constant should be about 5 milliseconds The field strength can be calculated as actual volts kV cuvette gap cm 15 8 Incubate the cells 3 hr at 30 C shaking at 250 rpm Plate aliquots of the electroporated cells on YM agar plates containing the appropriate selective media Incubate plates for 48 hrs at 30 C 7 3 Solutions and Reagents For Electroporation 1 YM broth 0 4 g yeast extract 10 g mannitol 0 1 g NaCl 0 1 g MgSO 0 5 g K HPO 3H 0 dissolve in 1 0 L of water and adjust to pH 7 0 Autoclave For YM plates add 15 g agar 1 L of YM broth
16. MicroPulser Electroporation Apparatus Operating Instructions and Applications Guide Catalog Number 165 2100 For Technical Service Call Your Local Bio Rad Office or in the U S Call 1 800 4BIORAD 1 800 424 6723 Section 1 1 1 1 2 1 3 Section 2 2 1 2 2 Section 3 3 1 3 2 3 3 Section 4 4 1 4 2 4 3 Section 5 5 1 5 2 5 3 Section 6 6 1 6 2 6 3 Section 7 7 1 7 2 7 3 Section 8 8 1 8 2 8 3 Section 9 9 1 9 2 9 3 Section 10 10 1 10 2 10 3 Section 11 11 1 11 2 11 3 Table of Contents Page Safety Information sessssesossesossesosscsosossosossesosoesosossosossosossesossesosossosossossssess 1 El ctrical Hazards tenno a aaie a Ea E E E a A 1 Mechanical Hazards 22 5 2 cccshaccbaesdeea aa aa 1 Other Safety Precautions cccececceesessessesessesesseseseeseeeseeeeseecesesseseeeeeeeaeeeeaeeeeaeeeeeees 1 Introducti nN sissicccssessscscasssossisecceessassesauaacisedsocscesssassisesieessonsseadiaesesnssensseasonsos 2 Overview of electroporation theory cceseeessesesseseeceseeseseeceseeseseeseeeseeeeseneeseeeenees 2 Manipulation of instrument parameters cceeeeceeeeeseeeeseeceseeceseeseeeseeeeseeeeseeeeseee 4 Factors Affecting Electroporation s sesessssessssesossosoesosossesosossosossesossesossee 5 Cellborowth i4 45cia ene haet thie tele EEEIEE EEEE ET EE EEE ET 6 IDNA EAE OAE AR S E A E E AE AEAEE te 6 Electroporation Media ccccecescssessesseseseesesee
17. NJ 209 1995 18 Leonardo E D and Sedivy J M A new vector for cloning large eukaryotic DNA segments in Escherichia coli Bio Technol 8 841 1990 19 Lin J J Electrotransformation of Agrobacterium in Methods in Molecular Biology 47 Nickoloff J A ed Humana Press Totowa NJ 171 1995 20 Miller E M and Nickoloff J A Escherichia coli electrotransformation in Methods in Molecular Biology 47 Nickoloff J A ed Humana Press Totowa NJ 105 1995 21 Nickoloff J A ed Methods in Molecular Biology 47 Humana Press Totowa NJ 1995 22 Park S F and Stewart G S A B High efficiency transformation of Listeria monocytogenes by electroporation of penicillin treated cells Gene 94 129 1990 23 Prentice H L High efficiency transformation of Schizosaccharomyces pombe by electroporation Nuc Acids Res 20 621 1991 24 Shigekawa K and Dower W J Electroporation of eukaryotes and prokaryotes A general approach to the introduction of macromolecules into cells Biotechniques 6 742 1988 21 25 26 27 28 29 30 31 32 33 Siguret V Ribba A S Cherel G Meyer D and Pieru G Effect of plasmid size on transfor mation efficiency by electrtoporation of Escherichia coli DH5a Biotechniques 16 422 1994 Simon D and Ferretti J J Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA FEMS Micobiol Lett 82 219 1991
18. agents For Electroporation 1 YPD 10 g yeast extract 20 g peptone dissolve in 900 ml water Autoclave Add 100 ml sterile 20 glucose 2 IM sorbitol 182 2 g sorbitol dissolve in 800 ml water Bring volume to 1 0 L with water Autoclave 3 20 glucose 20 g glucose dissolve in 60 ml water Adjust volume to 100 ml with water Sterilize through a 0 22 p filter Section 9 Electroporation of Schizosaccharomyces pombe 9 1 Preparation of Electrocompetent Cells See Prentice 1991 for additional information 1 Inoculate 500 ml of YCD in a 2 8 L Fernbach flask with an aliquot from an overnight culture of S pombe The doubling time of S pombe is approximately 2 hrs at 30 C 2 Incubate at 30 C overnight shaking at 250 rpm to a density of 1 x 107 cells ml OD o 0 7 3 Chill the cells in an ice water bath for 15 min to stop growth 4 Decant the cells into two sterile 250 ml centrifuge bottles and pellet the cells by centrifugation at 3000 x g for 5 min at 4 C 5 Carefully pour off and discard the supernatant place the centrifuge bottles with the cell pellets on ice 6 Add S0 ml of sterile ice cold 1 2 M sorbitol to each of the bottles and vortex to resuspend the cell pellets bring the volume in each of the centrifuge bottles to 250 ml Pellet the cells by centrifugation at 3000 x g for 5 min at 4 C pour off and discard the supernatant 7 Wash the cells again as in step 6 with a total of 250 ml sterile ice c
19. as a cryoprotectant for storage of cell cultures E NaCl 40u EB NaCl 200p o S a h MgCl2 40i 5008 A MgCl2 200 ui 500 0 0 2 4 6 8 10 Concentration mM B __ 3500 3000 0 PO4 6 1 404l ae PO4 6 1 200p g 2000 h PO4 7 3 40u E 1500 h P04 7 3 200l 1000 HEPES 7 5 40l E HEPES 7 5 200uI o 500 0 0 2 4 6 8 10 Concentration mM Fig 4 Resistance of solutions of A NaCl and MgCl2 and of B buffers of NaPO4 at pH 6 1 and 7 3 and HEPES at pH 7 5 Resistance was measured in 0 2 cm cuvettes containing either 40 pl or 200 ul of solution at room temperature Figures 4A and B show the effect of concentration of several biologically important ionic solutions on sample resistance Note that 1 volume has a significant effect on sample resistance for ionic solutions sample resistance is inversely proportional to the volume of solution in the cuvette 2 the resistance of a solution containing divalent ions is lower than a solution containing the same concentration of monovalent ions 3 the resistance of a buffered solution is affected by its pH The addition of even small concentrations of ionic compounds significantly reduces the resistance of the sample and may cause arcing Residual salt from ethanol precipitation of DNA should be reduced by washing the DNA pellet prior to dissolving it in either water or Tris EDTA Tab
20. buttons are pressed No pulse delivery the unit does not indicate PLS Pulse button is not depressed hard enough Verify instrument operation by the following tests Turn on the power switch Verify the LED next to Bacteria is illuminated Press the Settings button Verify the LED next to Fungi is illuminated Press the Settings button again Verify the LED next to Manual is illuminated Press the Settings button again The LED next to Bacteria should be lit Press the Raise button several times to change the display from Ec1 to Ec3 Press the Raise and Lower buttons simul taneaously to verify the display reads 3 00 volts Press the Pulse button until PLS is dis played a beep tone should be heard within 6 sec Press the Measurements button Verify the Actual kV LED illuminates Verify the display LED shows a reading between 2 95 and 3 05 Press the Measurements button again Verify the Time ms LED illuminates 22 Electrical Problem 1 Instrument shows Arc on display LED 2 Wrong time constant 3 Sample does not twitch 4 Instrument displays no on front panel 5 Instrument displays err on front panel Possible cause and solution Verify the display LED shows a reading between 5 50 and 6 50 If problems persists contact Bio Rad Arcing
21. ce is most noticeable in low resistance media These effects are discussed further in Section 3 3 The MicroPulser also includes a means to truncate the exponential decay pulse sooner than the expected time constant as long as the voltage is greater than 600 V When the pulse is terminated by the MicroPulser voltage is applied to the sample only for the specified time which may be between 1 0 and 4 0 msec Figure 3 shows how this waveform differs from the true exponential decay pulse Voltage V Time msec Fig 3 Truncation of an exponential decay pulse by the MicroPulser The solid line shows the volt age applied to the cells as a function of time during a pulse terminated after 2 5 msec The dashed line shows the voltage that would normally be applied to the cells during a true exponential decay pulse Section 3 Factors Affecting Electroporation The electrical conditions for the electroporation of microorganisms have been verified through years of research see Chang et al 1992 and Nickoloff 1995 for overviews as well as for protocols on electroporation of numerous species For many microorganisms opti mum electrotransformation occurs under electrical conditions relatively similar to those used for E coli and S cerevisiae two species that are most commonly used in research today For electroporation of E coli conditions reported as being used most often are 0 2 cm cuvettes con taining 40 wl of cells at a voltage of 2 5
22. ctroporation with cuvettes above 0 C reducing sample temperature increases sample resistance Samples electroporated in the Micropulser should have a time constant close to 5 msec If the time constant is much shorter than the expected value e g 3 msec instead of 5 msec the sample is too conductive The probable reasons for this are listed above under arcing Correct the problem of high conductivity by additional washing of the cells or by removal of salts from the DNA preparation This may mean that the pulse is not reaching the sample Check the connections between the MicroPulser and sample chamber Check to see that the contacts in the base of the sample chamber are not broken The manual setting is used and the voltage is set to 0 00 Use the Raise button to select a voltage between 0 02 and 3 00 kV Turn instrument off then on again If problem persists contact Bio Rad 23 Biological The general symptom addressed in this section is transformation efficiencies that are too low to detect or too low to be useful The following is a list of the areas of possible problems and some suggested solutions Problem Possible cause and solution 1 The pulse 2 The DNA 3 The cells 4 The temperature Is the pulse actually applied to the sample At high voltage with a small volume 40 ul sample this is easy to check The sample will twitch when pulsed If you don t see a twitch refe
23. d in ohms and the capacitance of the apparatus C is expressed in microfarads According to Equation 1 t is the time over which the voltage declines to 1 e 37 of the peak value The internal circuitry of the MicroPulser is designed to provide optimum electroporation of E coli and S cerevisiae as well as many other microorganisms in which the optimum transformation efficiency occurs at a time constant of approximately 5 msec These electroporation conditions are achieved by using a 10 microfarad capacitor and by placing a 600 ohm resistor in parallel with the sample cuvette along with a 30 ohm resistor in series with the sample cuvette Voltage V Time msec Fig 2 Exponential decay pulse from a capacitance discharge system When the capacitor charged to an initial voltage V is discharged into cells the voltage applied to the cells decreases over time so that at time t q the voltage is 1 e x V of the initial value In addition to the time constant the electric field strength is the other instrument param eter that is important in determining transformation efficiency The electric field strength E is the voltage applied between the electrodes and is described by E V d Equation 2 where V is the voltage applied and d is the distance cm between the electrodes The strength of the electric field and the size of the cells determine the voltage drop across each cell and it is this voltage drop that may be the imp
24. e 10 pg ml to 7 5 ug ml within this range the DNA concentration determines the probablility that a cell will be transformed At the higher DNA concentrations up to 80 of the survivors are transformed Dower et al 1988 Because the number of transformants recovered is the product of the transformation frequency and the number of cells present the transformation efficiency transformants ug DNA increases with cell concentration over the range of 10 to at least 3 x 10 cells ml Therefore to obtain a high transformation frequency use high DNA concentration To obtain high transformation efficiency use high cell concentration and low DNA concentration to avoid cotransformations In each case a small sample volume 20 50 ul allows economical use of DNA and cells see Dower et al 1988 for a detailed discussion of these factors 3 3 Electroporation Media The MicroPulser is designed for use with samples in high resistance media gt 600 ohms For this reason when preparing electrocompetent cells it is important to wash cells thoroughly to remove all traces of growth media Failure to thoroughly remove the growth media from the cells may result in the sample arcing during electroporation Cells should be washed at least three times with water or with non ionic solutions such as glucose glycerol sucrose sorbitol or polyethylene glycol For many microorganisms glycerol is a convenient electroporation medium since it is recommended
25. e components of the MicroPulser system and to Figure 5 for a definition of the buttons and LEDs 4 1 Setting Up The MicroPulser System 1 Connect the black power cord to the rear panel of the MicroPulser Pulse Generator Module Plug the cord into a wall outlet or power strip 2 Pull down the fold down foot on the underside of the MicroPulser Insert this foot into the track on the base of the shocking chamber Insert the shocking chamber slide into the shocking chamber 3 Connect the leads from the shocking chamber to the output jacks on the front panel of the MicroPulser polarity is not important to the electroporation process 4 Turn on the apparatus using the power switch on the right rear panel The light emitting diode LED display should illuminate and read Ec1 and the LED next to the Bacteria Settings should be illuminated 4 2 Operation of the MicroPulser 1 Selecting Pre Programmed Settings The MicroPulser is pre programmed with settings for electroporation of a number of commonly used organisms Included under the Bacteria Settings program are the following Mnemonic Organism Parameters Voltage Number of Time constant kV pulses msec Ect E coli 0 1 cm cuvette 1 8 1 Ec2 E coli 0 2 cm cuvette 2 5 1 StA S aureus 0 2 cm cuvette 1 8 1 2 5 Agr A tumefaciens 0 1 cm cuvette 2 2 1 Ec3 E coli 0 2 cm cuvette 3 0 1 Included under the Fungi Settings program are the following Mnemonic Organism Pa
26. ed When multiple pulses are delivered by one of the built in programs PLS is shown on the display LED during the entire time and a tone sounds each time a pulse is delivered To manually deliver multiple pulses after the tone sounds from the first pulse press the pulse button again Ifa lower pitched tone sounds accompanied by Arc being shown on the display LED the arc prevention and quenching ARQ system has been actuated and the pulse has been terminated This is usually an indication of attemped cuvette arc over but may also occur if the sample resistance is too low Since the energy delivered during such an ARQ event is low it is usually possible to pulse the sample again at parameters which will not result in an arc and still produce acceptable results However it is not advisable to use a sample in which two arc events have occurred 4 Measurements Pressing the Measurements button results in illumination of the Actual kV LED This indicates that the display LED shows the actual voltage delivered in kV during the last pulse If the instrument was just turned on and no pulse has been given the display LED shows 0 00 Pressing the Measurements button again results in illumination of the Time ms LED This indicates that the display LED shows the time constant in msec of the last pulse If the instrument was just turned on and no pulse has been given the display LED shows 0 00 Holding the Measurements button toggles
27. efficient than the corresponding circular plasmid in both E coli and Streptococcus pyogenes Shigekawa and Dower 1988 Simon and Ferretti 1991 Electroporation efficiency per mole of plasmid generally decreases as the plasmid size increases in numerous species including E coli Leonardo and Sedivy 1990 Siguret et al 1994 Pseudomonas aeruginosa Dennis and Sokol 1995 and Streptococcus thermophilus Somkuti and Steinberg 1988 However in some species including Lactococcus lactis Holo and Nes 1995 Enterococcus faecalis Cruz Rodz and Gilmore 1990 and Clostridium perfringens Allen and Blaschek 1990 transformation efficiency appears to be independent of plasmid size up to 20 30 kb Although transformation of most microorganisms has been accomplished using plasmid DNA isolated by a variety of methods the plasmid purity has an effect on transformation efficiency Significantly lower transformation efficiencies are generated with unpurified miniprep plasmid DNA than with plasmid DNA purified by a variety of procedures Plasmid produced using the Bio Rad Quantum matrix is as efficient as CsCl purified plasmid for transformation of microorganisms Generally for all types of microorganisms the frequency of transformation increases with inceasing DNA concentration in the electroporation buffer For coli the frequency of transformation transformants survivor is dependent on DNA concentration over at least six orders of magnitud
28. ells as close to 0 C as possible in an ice water bath and chill all containers in ice before adding cells To harvest transfer the cells to a cold centrifuge bottle and spin at 4000 x g for 15 minutes at 4 C Carefully pour off and discard the supernatant It is better to sacrifice the yield by pouring off a few cells than to leave any supernatant behind Gently resuspend the pellet in 500 ml of ice cold 10 glycerol Centrifuge at 4000 x g for 15 minutes at 4 C carefully pour off and discard the supernatant Resuspend the pellet in 250 ml of ice cold 10 glycerol Centrifuge at 4000 x g for 15 minutes at 4 C carefully pour off and discard the supernatant Resuspend the pellet in 20 ml of ice cold 10 glycerol Transfer to a 30 ml sterile Oakridge tube Centrifuge at 4000 x g for 15 minutes at 4 C carefully pour off and discard the supernatant Resuspend the cell pellet in a final volume of 1 2 ml of ice cold 10 glycerol The cell concentration should be about 1 3 x 1010 cells ml This suspension may be frozen in aliquots on dry ice and stored at 70 C The cells are stable for at least 6 months under these conditions 5 2 Electroporation 1 Thaw the cells on ice For each sample to be electroporated place a 1 5 ml microfuge tube and either a 0 1 or 0 2 cm electroporation cuvette on ice In a cold 1 5 ml polypropylene microfuge tube mix 40 ul of the cell suspension with 1 to 2 ul of DNA DNA should be in a low
29. ering Principles and Methods 12 Plenum Publishing Corp NY 275 1990 10 Dower W J Miller J F and Ragsdale C W High efficiency transformation of E coli by high voltage electroporation Nuc Acids Res 16 6127 1988 11 Dower W J Chassy B M Trevors J T and Blaschek H P Protocols for the transformation of bacteria by electroporation in Guide to Electroporation and Electrofusion Chang D C Chassy B M Saunders J A and Sowers A E eds Academic Press Inc 485 San Diego 1992 12 Heery D M and Dunican L K Improved efficiency M13 cloning using electroporation Nuc Acids Res 17 8006 1989 13 Holo H and Nes I F Transformation of Lactococcus by electroporation in Methods in Molecular Biology 47 Nickoloff J A ed Humana Press Totowa NJ 195 1995 14 Howard P K Ahern K G and Firtel R A Establishment of a transient expression system for Dictyostelium discoideum Nuc Acids Res 16 2613 1988 15 Jacobs M Wnendt S and Stahl U High efficiency electro transformation of Escherichia coli with DNA from ligation mixtures Nuc Acids Res 18 1653 1990 16 Knecht D and Pang K M Electroporation of Dictyostelium discoideum in Methods in Molecular Biology 47 Nickoloff J A ed Humana Press Totowa NJ 321 1995 17 Lee J C Electrotransformation of Staphylococci in Methods in Molecular Biology 47 Nickoloff J A ed Humana Press Totowa
30. fficiency increases as much as 60 fold from early to late log cultures Becker and Guarente 1991 The optimal portion of the growth phase to harvest cells is generally dependent on the cell type When preparing competent cells of a new species it is generally best to employ conditions worked out for use with the same genus Suggestions for factors to consider and general methods for producing electrocompetent cells are discussed in the articles by Dower et al 1992 and Trevors et al 1992 3 2 DNA While the majority of electroporation applications involve delivery of plasmid DNA to cells it should be mentioned that nearly any type of molecule can be introduced into cells by electroporation including RNA proteins carbohydrates and small molecules With few exceptions when delivering autonomously replicating plasmids the highest transformation efficiencies are obtained when electroporating supercoiled plasmid However electroporating plasmid that will integrate into the host genome is usually most efficient using linear plasmid For example Candida Pichia and Tetrahymena are transformed more efficently when transformed with linearized than with supercoiled integrating plasmids In both E coli and Listeria monocytogenes the transformation efficiency of relaxed circular plasmid is only slightly lower than that of supercoiled plasmid Leonardo and Sedivy 1990 Park and Stewart 1990 However linear plasmid is about 10 10 fold less
31. he cells by centrifugation at 400 x g for 5 7 min at room temperature Carefully pour off and discard the supernatant place the centrifuge bottles with the cell pellets on ice and resuspend the cells at a concentration of 1 x 10 cells ml Keep the cells on ice and use as soon as possible for electroporation 18 10 2 Electroporation 1 Pipette the DNA samples up to 50 ug to be electroporated into sterile 1 5 ml microfuge tubes Place tubes on ice Add 800 pl of the competent cells to each DNA sample and pipette up and down to mix incubate on ice 1 min Set the MicroPulser to dic See Section 4 for operating instructions Transfer the DNA cell samples to 0 4 cm electroporation cuvettes that have been chilled in ice and tap the suspension to the bottom of the tube Place the cuvette in the chamber slide Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber Pulse once the program delivers two pulses approximately 5 sec apart Remove the cuvette from the chamber and immediately dilute the cells to 10 ml with the appropriate media Check and record the pulse parameters The time constant should be millisecond The field strength can be calculated as actual volts kV cuvette gap cm When selecting for complementation of an auxotrophic mutant the cells may be plated immediately into selective media lacking the appropriate nutrient When selecting for antibiotic re
32. in the cuvette is the result either of an actual arc occurring or of medium that is too conductive An actual arc will occur usually only at high voltage gt 1500V If media is too conductive the display LED may show Arc even though an actual arc has not occurred The limit of Conductivity depends on the voltage electrode gap and sample volume but under standard conditions solutions of 10 meq or higher lt 600 ohms resistance will certainly indicate Arc To determine whether an actual arc has occurred or the media is too conductive look at the cuvette while pulsing the sample again If a small spark occurs across the electrodes an arc has occurred If no spark is observed lower the voltage and re pulse the sample Continue lowering and re pulsing the sample until a pulse occurs Check the time constant if the time constant is low lt 3 msec the sample is too conductive There are several causes of excessive conductivity 1 Washing and resuspending cells in a buffer too high in ionic strength 2 Insufficient washing of the cells salts from the growth medium are not completely removed the cells should be washed at least three to four times with non conductive solution 3 Lysed cells in the preparation cell contents contribute to conductivity 4 DNA solution too high in salt for example CsCl carried over from plasmid preparation or residual salts from ethanol precipitation or liga tion Ele
33. ionic strength buffer such as TE Mix well and incubate on ice for 1 minute Note it is best to mix the plasmids and cells in a microfuge tube since the narrow gap of the cuvettes prevents uniform mixing Set the MicroPulser to Ecl when using the 0 1 cm cuvettes Set it to Ec2 or Ec3 when using the 0 2 cm cuvettes See Section 4 for operating instructions 12 8 Transfer the mixture of cells and DNA to a cold electroporation cuvette and tap the suspension to the bottom Place the cuvette in the chamber slide Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber Pulse once Remove the cuvette from the chamber and immediately add 1 ml of SOC medium to the cuvette Quickly but gently resuspend the cells with a Pasteur pipette The period between applying the pulse and transferring the cells to outgrowth medium is crucial for recovering E coli transformants Dower et al 1988 Delaying this transfer by even 1 minute causes a 3 fold drop in transformation This decline continues to a 20 fold drop by 10 minutes Transfer the cell suspension to a 17 x 100 mm polypropylene tube and incubate at 37 C for 1 hour shaking at 225 rpm Check and record the pulse parameters The time constant should be close to 5 millisec onds The field strength can be calculated as actual volts kV cuvette gap cm Plate on selective medium 5 3 Solutions and Reagents For Electroporatio
34. iseconds 6 To turn the unit off press the power switch on the right rear panel The sample chamber may now be safely disconnected if desired Never remove the sample chamber cover until the leads are disconnected 11 Section 5 High Efficiency Electrotransformation of E coli Electroporation provides a method of transforming E coli at efficiencies as high as 10 to 10 transformants g which is greater than is possible with the best chemical methods The following protocol describes a method for for preparing and electrotransforming E coli to high efficiencies We are interested in hearing of additional strains transformed by electroporation and including this information in subsequent versions in our Electroprotocols manual Please contact your local Bio Rad representative access our web site at www bio rad com or in the U S call our Technical Services at 800 424 6723 with any comments or questions 5 1 Preparation of Electrocompetent Cells See Ausubel et al 1987 and Miller and Nickoloff 1995 for additional information Inoculate 500 ml of L broth with 1 100 volume of a fresh overnight E coli culture Grow the cells at 37 C shaking at 300 rpm to an OD of approximately 0 5 0 7 the best results are obtained with cells that are harvested at early to mid log phase the appropriate cell density therefore depends on the strain and growth conditions Chill cells on ice for 20 min For all subsequent steps keep the c
35. l and the cell concentration should be 1 x 10 cells ml Keep the cells on ice and use as soon as possible for electroporation 8 2 Electroporation 1 Pipette the DNA samples 5 100 ng in a volume of 5 ul to be electroporated into sterile 1 5 ml microfuge tubes Place tubes on ice 2 If0 2 cm cuvettes are used add 40 ul of the competent cells to each DNA sample if 0 4 cm cuvettes are used add 80 ul of the competent cells to each DNA sample Mix gently and incubate on ice for 5 min 3 Set the MicroPulser to Sc2 when using 0 2 cm cuvettes or to Sc4 when using 0 4 cm cuvettes See Section 4 for operating instructions 16 4 Transfer the DNA cell samples to the appropriate electroporation cuvettes that have been chilled in ice and tap the suspension to the bottom of the tube Place the cuvette in the chamber slide Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber Pulse once 5 Remove the cuvette from the chamber and immediately add 1 ml of ice cold 1 M sorbitol to the cuvette gently transfer the diluted cells into a sterile tube 6 Check and record the pulse parameters The time constant should be close to 5 milliseconds The field strength can be calculated as actual volts kV cuvette gap cm 7 Plate aliquots of the electroporated cells on selective agar plates containing M sorbitol Incubate plates for 48 72 hrs at 30 C 8 3 Solutions and Re
36. le shows that although adding a solution of plasmid in 10 mM Tris pH 8 0 1 mM EDTA to water does reduce the sample resistance this should not result in the inability to electroporate a sample in the MicroPulser DNA may be used directly from enzyme reactions for transformation but the final salt concentration in the electroporation sample should be kept below 5 meq for high voltage operation Finally ligation mixtures may be used for transformation but only in very low quantities or when the ionic strength is reduced by dilution Willson and Gough 1988 dialysis Heery and Dunican 1989 Jacobs et al 1990 or ethanol precipitation B ttger 1988 Zabarovsky and Winberg 1990 Table 1 Resistance of Water in 0 2 cm Cuvettes To Which TE Has Been Added SAMPLE sample Roimpi 40 ul volume 200 ul volume Water gt 6x 10 gt 6x 10 Water 1 ul TE gt 6x 10 35 000 Water 5 u TE 11 200 8 700 Water 10 ul TE 4 850 4 700 The resistance of 0 2 cm cuvettes containing either 40 or 200 ul water and the indicated volume of TE 10 mM Tris pH 8 0 1 mM EDTA was measured at 1000 V MicroPulser Settings Measurements Bacteria Actual kV on Ww Time amp ms Manual Raise Display LED Settings Measurements Pulse and Settings button button button Lower LEDs Measurements Buttons LEDs Fig 5 MicroPulser control panel Section 4 MicroPulser Operating Instructions Refer to Figure 1 for a view of th
37. meters The time constant should be close to 2 5 milliseconds The field strength can be calculated as actual volts kV cuvette gap cm Plate aliquots of the electroporated cells on trypticase soy agar containing selective antibiotic Incubate plates for 36 48 hrs at 37 C 6 3 Solutions and Reagents For Electroporation I B2 medium 10 g casein hydrolysate 25 g yeast extract 5 g glucose 25 g NaCl 1 g K HPO dissolve in 900 ml water and adjust pH to 7 5 bring volume to 1 0 L Autoclave SMMP 55 ml 2X SMM 40 ml 4X Penassay broth 5 ml 10 w v bovine albumin adjust pH to 7 0 filter sterilize Trypticase soy agar 40 g trypticase soy agar Becton Dickinson Sparks MD in 1 L of water Autoclave 2X SMM 25 ml 0 2 M sodium hydrogen maleate 40 ml 0 1 N NaOH adjust the pH to 6 5 Add 5 ml 1 M MgCl 42 7 g sucrose dissolve and bring volume to 125 ml Filter sterilize 4X Penassay broth 17 5 g Antibiotic Medium 3 Becton Dickinson dissolved in 250 ml water Autoclave 0 2 M sodium hydrogen maleate 11 6 g maleic anhydride or 13 7 g maleic acid 4 g NaOH dissolve in 500 ml water Autoclave 14 Section 7 Electroporation of Agrobacterium tumefaciens 7 1 Preparation of Electrocompetent Cells See Lin 1995 for additional information 1 Inoculate 1 5 L of YM broth in a 2 8 L Fernbach flask with an aliquot from log phase culture of A tumefaciens 2 Incubate at 30 C overnight shaking at 300 rpm to a densit
38. n 1 L Broth 10 g Bacto tryptone 5 g Bacto yeast extract 5 g NaCl dissolve in 1 0 L water Autoclave 2 10 v v Glycerol 12 6 g glycerol density 1 26 g cc in 90 ml of water Autoclave or filter sterilize 3 TE 10 mM Tris HCl pH 8 0 1 mM EDTA 4 SOC 2 Bacto tryptone 0 5 Bacto yeast extract 10 mM NaCl 2 5 mM KCl 10 mM MgCl2 10 mM MgSO4 20 mM glucose Section 6 Electroporation of Staphylococcus aureus 6 1 Preparation of Electrocompetent Cells See Lee 1995 for additional information Inoculate 3 ml of B2 broth in a 17 x 100 mm tube with a colony from a fresh S aureus plate Incubate at 37 C overnight shaking at 250 rpm Inoculate 1 5 ml of the overnight culture into 150 ml of fresh B2 broth in a 1 liter flask Incubate at 37 C shaking at 250 rpm to 2 x 108 cells ml The doubling time of S aureus is about 30 min at 37 C Chill the cells in an ice water bath for 15 min to stop growth Decant the cells into a sterile 500 ml centrifuge bottle Harvest the cells by centrifugation at 12 000 x g for 15 min at 4 C Carefully pipette off the supernatant keeping the cell pellet on ice Resuspend the cell pellet in 500 ml of sterile ice cold water Pellet the cells by centrifugation at 12 000 x g for 15 min at 4 C carefully remove the supernatant Wash the cells 2 more times in 500 ml of sterile ice cold water 13 Resuspend the cell pellet in 25 ml of sterile ice cold 10 glycerol Transfer
39. o Rad Laboratories Website www bio rad com Bio Rad Laboratories Main Office 2000 Alfred Nobel Drive Hercules CA 94547 Ph 510 741 1000 Fx 510 741 5800 Also in Australia Ph 02 9914 2800 Fx 02 9914 2889 Austria Ph 01 877 89 01 Fx 01 876 56 29 Belgium Ph 09 385 55 11 Fx 09 385 65 54 Canada Ph 905 712 2771 Fx 905 712 2990 China Ph 86 10 62051850 51 Fx 86 10 62051876 Denmark Ph 45 39 17 99 47 Fx 45 39 27 16 98 Finland Ph 358 0 9 804 2200 Fx 358 0 9 804 1100 France Ph 01 43 90 46 90 Fx 01 46 71 24 67 Germany Ph 089 318 84 0 Fx 089 318 84 100 Hong Kong Ph 852 2789 3300 Fx 852 2789 1257 India Ph 91 11 461 0103 Fx 91 11 461 0765 Israel Ph 03 951 4127 Fx 03 951 4129 Italy Ph 39 02 216091 Fx 39 02 21609 399 Japan Ph 03 5811 6270 Fx 03 5811 6272 Korea Ph 82 2 3473 4460 Fx 82 2 3472 7003 Latin America Ph 305 894 5950 Fx 305 894 5960 Mexico Ph 514 2210 Fx 514 2209 The Netherlands Ph 0318 540666 Fx 0318 542216 New Zealand Ph 64 9 4152280 Fx 64 9 4152284 Norway Ph 22 74 18 70 Fx 22 74 18 71 Russia Ph 7 095 979 98 00 Fx 7 095 979 98 56 Singapore Ph 65 2729877 Fx 65 2734835 Spain Ph 34 91 661 7085 Fx 34 91 661 9698 Sweden Ph 46 0 8 55 51 27 00 Fx 46 0 8 55 51 27 80 Switzerland Ph 01 809 55 55 Fx 01 809 55 00 United Kingdom Ph 0800 181134 Fx 01442 259118 Sig 031799 4006174 Rev B
40. old 1 2 M sorbitol 8 Resuspend the cell pellet in 20 ml of sterile ice cold 1 2 M sorbitol and transfer to a chilled 30 ml Oakridge tube Pellet the cells by centrifugation at 3000 x g for 5 min at 4 C pour off and discard the supernatant 9 Resuspend the cell pellet in 0 5 ml of sterile ice cold 1 2 M sorbitol the final cell volume should be 1 3 ml and the cell concentration should be 1 x 10 cells ml Keep the cells on ice and use as soon as possible for electroporation 17 9 2 Electroporation 1 Pipette the DNA samples up to 1 ug to be electroporated into sterile 1 5 ml microfuge tubes Place tubes on ice Add 200 ul of the competent cells to each DNA sample and mix gently Set the MicroPulser to ScS See Section 4 for operating instructions Transfer the DNA cell samples to 0 2 cm electroporation cuvettes that have been chilled in ice and tap the suspension to the bottom of the tube Place the cuvette in the chamber slide Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber Pulse once Remove the cuvette from the chamber and immediately add 0 8 ml of ice cold 1 2 M sorbitol to the cuvette gently transfer the diluted cells to a sterile tube Check and record the pulse parameters The time constant should be close to 5 milliseconds The field strength can be calculated as actual volts kV cuvette gap cm Incubate the tubes at room temperature for 40 6
41. ortant manifestation of the voltage effect in elec troporation The purpose of the 30 ohm series resistor in the MicroPulser is to protect the instrument circuitry should arcing occur Under normal operation when samples are in high resistance media this resistor will not affect the voltage applied to the sample However this resistor will significantly decrease the voltage applied to the sample if the resistance of the sample is low The fractional drop in voltage applied to the sample is given by Ryo Ryo R ample When R sie 18 600 ohms there is a 5 voltage drop to the sample 30 30 600 0 048 For this reason electroporation with the MicroPulser should not be performed in solutions with a resistance of less than 600 ohms This includes samples in which the growth medium was not adequately removed from the cells DNA samples containing salt contributed by residual sodium chloride or ligation mixtures The MicroPulser is able to measure the resistance of the sample and will not pulse into very low resistance media 2 2 Manipulation of Instrument Parameters Several parameters on the MicroPulser may be altered to achieve maximum transformation efficiency These include the field strength E the time constant t and the width of a truncated exponential decay pulse The field strength may be manipulated in two ways First voltages between 200 and 3000 V may be set directly on the MicroPulser This parameter is the most easil
42. r to the electrical troubleshooting section for information on electrical problems Also make sure that the cuvette is making contact with the electrodes at the back of the sample chamber If electrodes are broken or corroded call Bio Rad for replacements Are the amplitude and length of the pulse suffi cient E coli requires pulses of approximately 5 msec with field strengths of 12 to 18 kV cm S cerevisiae requires pulses of approximately 5 msec with a field strength of 7 5 kV cm There is usually some cell death with electrical conditions producing transformation Survival rates of 20 to 80 are typical If no cell death occurs the pulse is probably too weak Conversely if too many cells are killed gt 80 the pulse is probably too intense and transformation will probably be poor To find the optimum pulse characteristics use a pulse length of 5 msec and test for transformation over a range of field strengths Check the quantity and quality of the DNA on a gel Often mini preps contain less DNA than expected DNA stored improperly for long periods may be degraded and lack transforming activity Some preparations of DNA may contain substances that inhibit transformation or are toxic to the cells Try to use DNA free of SDS phenol etc Is the selection appropriate for the marker and its level of expression Were the cells harvested at the correct stage in the growth phase Bacterial cells generally show the highest tran
43. rameters Voltage Number of Time constant kV pulses msec Sc2 S cerevisiae 0 2 cm cuvette 1 5 1 Sc4 S cerevisiae 0 2 cm cuvette 3 0 1 ShS S pombe 0 2 cm cuvette 2 0 1 dic D discoideum 0 4 cm cuvette 1 0 2 1 0 Pic P pastoris 0 2 cm cuvette 2 0 1 Pressing the Settings button cycles the Settings LED between Bacteria Fungi and Manual When the LED next to Fungi is lit the mnemonic for the fungi programs are displayed Pressing the Raise and Lower buttons to the left of the display LED cycles between the different fungi programs When the mnemonic is displayed the parameters associated with the mnemonic are automatically selected To change from one bacteria setting to another while the Settings LED next to Bacteria is lit press the Raise and Lower buttons to the left of the display LED to cycle between the different programs When the mnemonic is displayed the parameters associated with the mnemonic are automatically selected While a program mnemonic is displayed for either a Bacteria or Fungi Setting simultaneously pressing both the Raise and Lower buttons shows the program parameters selected on the display LED The display LED first shows the voltage value then displays a t followed by the time in msec then if time and multiple pulses are associated with a program display P followed by 2 indicating that two successive pulses are given If no t is given the pulse is not truncated
44. seseeseseeseseeseeeeseeeeseeeeseeeeeeeseeeeseeeeseeeeeees 7 MicroPulser Operating InstructionS sesessesesossesoesesossesossssesossosossesossse 9 Setting up the MicroPulser System s sessssssssesesesesesesessssesesesesestsesesesesessssesesesesesese 9 Operation of the MicroPulser c ccceceseesessesesseseseeseeeeseseseeceseeeeseeeeeeseeeeseeeeseeeeeees 9 Electroporation using the MicroPulser ccceccsceeeseseeseseeseseeeeeeeseeeseeeeseeeeseeeees 11 High Efficiency Electrotransformation Of E Coli s csccscssssssscseeoesees 12 Preparation of electrocompetent Cells cceseeesssseseeseseeeseeeeeeeseeseseeseseeseseseeeeeaees 12 Electroporation EEEE E A E E ia eee ie eee na aa T awn 12 Solutions and reagents for electroporation ccceceseeseseeseseeseseeceeeeseeeeseeseseeeeeeeees 13 Electroporation of StaphylocOCCUs AUF eUS sessssessssesossosossososossosossosossesosoe 13 Preparation of electrocompetent Cells cceceeesseseseeseseeseeeseeeeseeeeseeeeseeeeseeeeeeeaees 13 Electroporation ccccccccecesesceseeseseeseseeceseeseseesesecseeeseeeesecsesecaeeeseeesseesesecaeeeeaeeeeaeeaeas 14 Solutions and reagents for electroporation ccceceseeseseeseseeseseeseeeeseeseseeeeseeeeseees 14 Electroporation of Agrobacterium tumefaciens sssssssssossssosossesossosossesosoe 15 Preparation of electrocompetent Cells ceseeesesseseeseseeseseeeeeeeseeeseescseeeeseseeeeenees 15 El ectropora
45. sformation efficiencies when harvested in the early to mid log growth phase Yeast cells generally show the highest transformation effiencies when harvested in late log phase Different growth conditions may improve transformation Are too many cells killed The pulse is too intense toxic substances are present in DNA or cell preparations wrong temperature of electroporation are all possibilities Are the cells transferred to outgrowth medium immediately after the pulse For E coli this is very important Is the correct selection applied after the recovery period Are the cuvettes cold Is the cuvette holder slide prechilled If frozen have the cells been stored properly usually in 10 15 glycerol at 70 C 24 Appendix III Product Information Specifications Input voltage Input current Maximum output voltage and current Output waveform Output voltage adjustment Ambient operating temperature Dimensions H x W x D Weight Related products 100 120 V RMS 50 60 Hz automatic mains voltage switching 220 240 V RMS 50 60 Hz 2 amp RMS 100 120 V 1 amp RMS 220 240 V 3000 V peak into gt 3 3 kohm load limited to 100 amp peak maximum Decaying exponential waveform with RC time constant of 5 msec assuming loads of 3 3 kohm Voltage adjustable in 200 3000 V range with 10 V display resolution 10 pre programmed voltage steps 3 5 35 C 8x 21x 31cm 2 9 kg 6 4 Ibs Cuvettes Catalog
46. sistance incubate the cells overnight at 21 C prior to adding the selective agent 10 3 Solutions and Reagents For Electroporation l HL5 media 17 8 g bacteriological peptone Oxoid Ogdensburg NY 7 2 g yeast extract 0 54 g Na HPO 0 4 g KH PO 130 wl B12 Folic acid mix bring to 1L with water and adjust to pH 6 3 6 5 Autoclave for 25 min on two successive days Prior to use add 20 ml of 50 glucose and 10 ml of 100 X Antibiotic Antimycotic Life Technologies Gaithersburg MD B12 Folic acid mix 5 mg B12 200 mg folic acid add 95 ml water then pH to 6 5 6 8 with SN NaOH bring to 100 ml with water Filter sterilize and store at 20 C protected from light E buffer 10 ml 100 mM NaH PO adjusted to pH 6 1 with KOH 10 ml 0 5 M sucrose 80 ml water autoclave Section 11 Electroporation of Pichia pastoris 11 1 Preparation of Eelectrocompetent Cells See Cregg amp Russell 1998 for additional information Inoculate 500 ml of YPD in a 2 8 L Fernbach flask with an aliquot from a fresh overnight culture of P pastoris The doubling time of P pastoris is approximately 2 hrs at 30 C Incubate at 30 C overnight shaking at 300 rpm to a density of 5 7 x 10 cells ml Decant the cells into two sterile 250 ml centrifuge bottles and pellet the cells by cen trifugation at 3000 x g for 5 min at 4 C Carefully pour off and discard the supernatant 19 Add 50 ml of sterile YPD HEPES to each of the bottles
47. tionia scsint an E E T R E RAN 15 Solutions and reagents for electroporation se ssseseesssesesesesesestsrserersesesesesesesesesees 16 Electroporation of Saccharomyces CereviSide s scrscescsrersresccseccecrsees 16 Preparation of electrocompetent Cells s ssssssseseseseseseseeseresesesesesesrsrseeeseseseseseses 16 Electtoporations snorri ie se cucses E E E E soe escneeis E E E E ERRE 16 Solutions and reagents for electroporation s essssessesesesesesesestseserersesesesesesesesesess 17 Electroporation of Schizosaccharomyces pomMbe ssssssssssssessssesossosossososos 17 Preparation of electrocompetent cells s sssssesesesesesesesereesesesssesesesesrseeesereseseseses 17 Electroporation e e a E ER E EE ERE 18 Solutions and reagents for electroporation ecceceseeseseeseseeseseeceeeeseeseseeeeseeeeseeees 18 Electroporation of Dictyostelium discoideum sssessesrrssesccseseecrsceseees 18 Preparation of electrocompetent Cells ceseeesesseseeseeeseseeceeeseeseseeeeseeeeseeeeerenens 18 Electroporation oxox exe ccezcs sn cng ce ancy ta toc Dea cds As a ARR AEA RER A ERE 19 Solutions and reagents for electroporation cccecesseseseeseseeseeeeseeeeseeseseereseeeeeeeees 19 Electroporation Of Pichia pastoris esssssssssssesossesossosossosossososossesossososoesosoe 19 Preparation of electrocompetent cells s sssssseseseseseseseseeeesesesesesesesrseseeeseseseseseses 19 Electroporation
48. to a 30 ml sterile Oakridge tube Pellet the cells by centrifugation at 12 000 x g for 15 min at 4 C carefully remove the supernatant Resuspend the cell pellet in 2 ml of 10 glycerol the final cell concentration should be 1 x 1010 cells ml Dispense 250 ul aliquots of the electrocompetent cells into sterile 1 5 ml microfuge tubes freeze the cells in an isopropanol dry ice bath then store at 70 C The cells are stable for several months under these conditions 6 2 Electroporation 1 Pipette the DNA samples 5 ng 2 ug in a volume 3 ul to be electroporated into sterile 1 5 ml microfuge tubes Thaw the competent cells at room temperature for several minutes Add 50 ul of cells to each DNA sample gently pipette up and down to mix Incubate the samples at room temperature for 30 min Set the MicroPulser to StA See Section 4 for operating instructions Transfer the mixture of cells and DNA to a 0 2 cm electroporation cuvette and tap the suspension to the bottom of the tube Place the cuvette in the chamber slide Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber Pulse once Remove the cuvette from the chamber and immediately add 1 ml of SMMP medium containing a subinhibitory concentration of antibiotic gently transfer the cells to a sterile 17 x 100 mm tube using a Pasteur pipette Incubate 1 hr at 37 C shaking at 250 rpm Check and record the pulse para
49. y controlled The process of varying the voltage while keeping all other conditions unchanged is the basis for most electroporation optimization procedures Second using cuvettes with different electrode gap widths permits a means of changing the field strength For electroporation of microorganisms 0 1 and 0 2 cm gap cuvettes are most often used Electroporation of E coli is generally carried out at a voltage of 1 8 kV E 18 kV cm when electroporating cells in 0 1 cm cuvettes and at a voltage of 2 5 kV E 12 5 kV cm when electroporating cells in 0 2 cm cuvettes These electroporation conditions are pre programmed into the MicroPulser as programs Ecl V 1 8 kV and Ec2 V 2 5 kV in the bacterial settings menu In addition a third program Ec3 in the bacterial settings menu delivers a voltage of 3 0 kV E 15 kV cm in 0 2 cm cuvettes which we have found results in even higher transformation efficiency compared to electroporation at 2 5 kV The time constant may be altered by changing the sample resistance The sample resistance may be manipulated in two ways First increasing the salt or buffer concentration of the electroporation media decreases the resistance of the sample and vice versa resulting in a change in the time constant Second the volume of the sample in the cuvette is inversely proportional to the resistance of the sample decreasing the sample volume increases the sample resistance This effect of volume on sample resistan
50. y of 5 10 x 10 cells ml 3 Decant the cells into sterile 500 ml centrifuge bottles and pellet the cells by centrifugation at 3000 x g for 10 min at 4 C 4 Carefully pour off and discard the supernatant place the centrifuge bottles with the cell pellets on ice 5 Add 50 ml of sterile ice cold 10 glycerol to each of the bottles and vortex to resuspend the cell pellets bring the volume in each of the centrifuge bottles to 500 ml with sterile ice cold 10 glycerol Pellet the cells by centrifugation at 3000 x g for 10 min at 4 C pour off and discard the supernatant 6 Wash the cells again as in step 5 7 Resuspend each of the cell pellets in 5 ml of sterile ice cold 10 glycerol and transfer to a chilled 30 ml Oakridge tube Pellet the cells by centrifugation at 3000 x g for 5 min at 4 C pour off and discard the supernatant 8 Resuspend the cell pellet in 0 5 ml of sterile ice cold 1 M sorbitol the final cell volume should be 1 5 ml and the cell concentration should be 5 x 1010 cells ml Dispense 200 ul aliquots of the electrocompetent cells in sterile 1 5 ml microfuge tubes freeze the cells in an isopropanol dry ice bath then store at 70 C The cells are stable for about 6 months under these conditions 7 2 Electroporation 1 Pipette the DNA samples up to 5 ul to be electroporated into sterile 1 5 ml microfuge tubes the DNA should be in either water or TE Place tubes on ice 2 For each DN
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