E Coli Pulser User Manual
Contents
1. 14 5 2 2 Optimizing Electrotransformation Efficiency of E coli Cells 14 5 3 Medium and Buffer Formulations AEL 15 65 Referente DD TM 16 Wele e UR D Ul 17 8 Additional Information 2 2 22 2 42 000000000000 18 8 1 Care and 22 2 18 8 2 Maintenance een 18 8 3 e Ede ec pe eg dat 19 SA aa eee Cae dia 20 8 5 Equipment Decontamination Certificate 21 8 0 22 8 7 Declaration of Conformity and CE 222 2 2 22 Table of Contents Figures 1 CELL PORATOR E coli Electroporation System EE 2 2 Evcoli Pulser Front Control sete reed b YR pe dayik Han 3 3 Single Safe and Disposable Microelectroporation 4 4 Basic Design of an Electroporation System 5 Capacitor Discharge 2 6 Schematic Diagram of CELL PORATOR E coli System Pulse Circuit 7 7 CELL PORATOR E coli System Pulse Shape 22 2 2 8 7 Table Expected Electrotransformation Efficiencies of E coli Strains with pUCTO DNA si u RE 13 CELL PORATOR and HORIZON are registered trademarks of Whatman DELRIN is a regi2. and Postmes A M F A 1987 Nucleic Acids Res 15 3629 Zeugin J and Harley J 1985 Focus 7 4 1 Crouse J and Amorese D 1987 Focus 9 2 3 Related Products Product Specification Accessories E coli Pulser USA Single Safe 1 chamber capacity Chamber Safe 4 chamber capacity Related Products CeLL PORATOR Electroporation USA System with 0 4 cm gap disposable electroporation chambers CELL PORATOR Electroporation USA System with 0 15 cm gap disposable microelectroporation chambers Chamber Rack 0 4 gap Disposable Electroporation pkg of 50 Electroporation Chambers 0 15 cm gap Disposable pkg of 50 Microelectroporation Chambers CELL PORATOR Voltage Booster USA Pulse Control USA CAN Cat No 11613049 11613064 11602018 71600019 11609013 11606019 11601028 11608031 11612017 11604022 Additional Information 8 1 Care and Handling The components of the CELL PORATOR E coli Electroporation System are fabricated from steel aluminum and D amp RIN As with any laboratory instrument adequate care ensures consistent and reliable performance The external surfaces of the components may be cleaned with a damp cloth or paper towel and mild detergent To remove grease and oils use a light application of hexane kerosene or aliphatic naphtha Never use abra sive cleaners window sprays or rough cloths to clean the components as these can cause surface damage Additio
3. Base contains temperature control compartment Figure 3 Single Safe and Disposable Microelectroporation Chambers Important Each disposable microelectroporation chamber should be used only once for reliable and consistent results Autoclaving distorts the chambers and does not remove or destroy residual DNA or other material in the crevices between chamber walls and elec trodes Agents such as bleach that destroy DNA and other organic compounds will dam age the aluminum electrodes and alter their electrical properties For purposes of this manual all CELL PORATOR E coli Electroporation System components should be used only in conjunction with one another as instructed However certain alternative configurations may also be possible using other CELL PORATOR products that are not part of this system e g E coli pulser with chamber safe chamber rack and pulse cable single safe with pulse control and voltage booster please call the Whatman EEES Service Department or Technical Support see Chapter 8 4 for numbers for additional details and precautions Warning Under no circumstances should any CELL PORATOR product be used conjunc tion with any other type of electroporation apparatus from other manufacturers or any other laboratory equipment 2 3 Introduction to Electroporation 2 3 1 Background Information Electroporation is a technique for rendering cell membranes temporarily permeable to macromolecules such as DNA
4. Gregg R G Boggs S S Koralewski M A and Kucherlapati R S 1985 Nature Lond 317 230 Reiss M Jastreboff M M Bertino J R and Narayanan R 1986 Biochem Biophys Res Commun 137 244 10 Toneguzzo F Hayday A C and Keating A 1986 Mol Cell Biol 6 703 Tur Kaspa R Teicher L Levine B J Skoultchi A l and Shafritz D A 1986 Mol Cell Biol 6 716 12 Fromm M Taylor L P and Walbot V 1986 Nature Lond 319 791 Bates G 1986 unpublished observations 14 Nishiguchi M Langridge W H R Szalay A A and Zaitlin M 1986 Plant Cell Rep 5 57 15 Okada K Nagata T and Takebe 1986 Plant Cell Physiol 27 619 16 Sowers A S and Lieber M R 1986 FEBS Lett 205 179 17 Sokoloski J A Jastreboff M M Bertino J R Sartorelli A C and Narayanan R 1986 Anal Biochem 158 272 18 Smith M Jessee J Landers T and Jordan J 1990 Focus 12 38 19 Dower W J Miller J F and Ragsdale C W 1988 Nucleic Acids Res 16 6127 20 21 22 23 24 Calvin N M and Hanawalt P C 1988 J Bacteriol 170 2796 Hanahan D Jessee J and Bloom F 1991 Methods Enzymol 204 63 Hanahan D 1983 J Mol Biol 166 557 Lorow D and Jessee J 1990 Focus 12 19 Grant S Jessee J Bloom F and Hanahan D 1990 Proc Natl Acad Sci USA 87 4645 Upcroft P and Healy A 1987 Gene 51 69 Dorssers
5. col Place cells DNA and the required number of sterilized 1 5 ml microcentrifuge tubes on ice Remove the required number of disposable microelectroporation chambers from their plastic pouches and place them on ice Note At this and all subsequent steps take care to avoid contaminating the cham bers during handling In sterilized microcentrifuge tubes prepare the cell DNA mixture to be electroporat ed For additional information please refer to Chapter 5 Once sample preparation is complete repeat steps 2 through 7 of the preceding protocol Open a disposable microelectroporation chamber gripping the sides of the cap Hold back the cap of the chamber with your index finger and using a micropipette pipet 20 of the cell DNA mixture between the bosses of the chamber Apply the sample slowly first making contact with one boss and then drawing the sample over to the other Note We recommend practicing the loading technique with 20 ul of a sterilized 2096 solution of glycerol in water before attempting to load critical samples Close the cap of the disposable microelectroporation chamber and gently place the loaded chamber into the single safe base Orient the single safe lid over the base using the positioning post and electrode rivets see figure 3 Tighten the threaded outer ring to secure the lid in place for electroporation Note Be careful not to crossthread the outer ring poor electrical contact and loss of the sample may
6. is shown in figure 7 For 20 samples suspended 10 glycerol in water the pulse length at all three VOLT AGE RANGE settings is 10 ms Volts Time Figure 7 CELL PORATOR E coli System Pulse Shape Operating Instructions 3 1 Procedural Overview The basic steps of an electroporation experiment using the CELL PORATOR E coli Electro poration System are as follows 1 Render cells electrotransformable see Chapter 5 or purchase electrotransformable cells Add DNA to cells Electroporate Transfer the cells into the appropriate medium E CE Select for transformants 3 2 Setting Up the Work Area If possible the CELL PORATOR E coli Electroporation System should be set up for operation in or at least close to a laminar flow hood which allows transfer of cells and media to and from the disposable microelectroporation chambers under reasonably sterile condi tions To this end the CELL PORATOR E coli Electroporation System is designed to be trans ported easily and to occupy minimal bench space 3 3 Setup for Temperature Control The base of the single safe is a thermal mass that may be used to help maintain the desired sample temperature of 4 for electroporation Once the following steps have been completed the base may be kept indefinitely gt 4 C refrigeration or on ice being removed to room temperature only for the duration of setting up and performing the electroporation rea
7. result Press the CHARGE button to the capacitor Note When the unit is ready to deliver a pulse the READY indicator light on the front panel will be lit With the VOLTAGE RANGE selector switch set to MEDIUM the LCD voltmeter should display a reading of 2 5 kV Confirm that the READY indicator light is lit and push the TRIGGER button to deliver the pulse to the sample Note tone sounds to indicate that the voltage is delivered and the LCD voltmeter reading should drop to 0 Loosen the threaded outer ring and remove the single safe lid Gently lift out the dis posable microelectroporation chamber for further processing of the sample After removing the disposable microelectroporation chamber from the single safe carefully open the cap Using a micropipette remove the sample from the electrode bosses and place into a culture tube containing 1 ml of S O C medium from step 1 of this protocol Express samples for 1 h in an incubator shaker at 37 225 rpm Plate the cells on selective medium and select for transformants For additional samples repeat steps 5 through 12 Once the experiment is finished check the voltmeter to make sure the capacitor is discharged the LCD voltmeter should read 0 and turn the POWER switch on the E coli pulser to the off position Disconnect the E coli pulser from the AC outlet Return the single safe base to cold storage 4 until needed Use a damp cloth or paper
8. towel to wipe off any spills from the instruments Store the components in a dry noncorrosive environment 10 Troubleshooting Guide Many apparatus problems can be solved by carefully following the instructions in this manual Some suggestions for troubleshoot ing are given in this chapter Should these suggestions not resolve the problem call the Whatman Service Depart ment or Technical Support see Chapter 8 4 for numbers If the unit must be returned for repair contact our service department the technical support or your local distributor for shipping instructions Include a full description of the problem Problem Equipment The POWER indicator light does not come on and the LCD voltmeter shows no reading The POWER indicator light does not come on but the LCD voltmeter shows a reading The POWER indicator light comes on but the LCD voltmeter shows no reading After pressing the CHARGE button the LCD voltmeter shows no increase in voltage and the READY light does not come on After pressing the CHARGE button the LCD voltmeter displays the expected value but the READY light does not come on Nothing happens when the pulse is triggered no tone heard no change on the LCD voltmeter A loud crack is heard at pulse discharge The temperature control compartment is leaking fluid Comment Verify that the POWER switch is set to the on I position Verify that the AC power cord is securely plugge
9. 0 x g Remove the supernatant with a micropipette 3 Add 60 ul of 7096 ethanol and centrifuge for 15 min Remove the supernatant with a micropipette 4 Dry the pellet briefly under a vacuum 5 Using micropipette resuspend the pellet in 20 ul of 0 5x TE buffer To avoid con tact with any salts dried further up on the side of the tube wet only the part of the tube immediately around the pellet 6 Transfer the resuspended DNA to a new microcentrifuge tube and add to cells as specified in Section 5 1 5 2 2 Optimizing Electrotransformation Efficiency of E coli Cells VOLTAGE RANGE Setting The CELL PORATOR E coli System is designed to optimize these parameters for electroporation of E coli cells With the VOLTAGE RANGE selector switch on the E coli pulser set to MEDIUM 2 5 kV the fixed capaci tance of 2 uF and internal parallel resistance of 4 E coli cell DNA mixtures prepared and electroporated as described should yield consistently high transfor mation efficiencies Although field strength may be varied by turning the VOLTAGE RANGE selector switch to the LOW or HIGH setting this step should be performed only following analysis of transformation efficiencies obtained at the MEDIUM set ting see Chapter 4 Cell Density Because the resistance of the circuit is largely determined by the sam ple varying the density of the cell suspension may affect transformation efficiency For E coli the optimal cell density
10. Clean all parts of the instrument from residues and of biologically dangerous chemical and radioactive contaminants Please include a written confirmation use the Equipment Decontamination Certificate following on the next page that the device is free of biologically dangerous and radioactive contaminants in each shipment If the device is contaminated it is possible that Biometra will be forced to refuse to accept the device The sender of the repair order will be held liable for possible damages resulting from insufficient decontamination of the device Please enclose a note which contains the following a Sender s name and address b Name of a contact person for further inquiries with telephone number 20 8 5 Equipment Decontamination Certificate To enable us to comply with german law i e 871 StrlSchV 817 GefStoffV and 819 ChemG and to avoid exposure to hazardous materials during handling or repair will you please complete this form prior to the equipment leaving your laboratory COMPANY INSTITUTE ADDRESS TEL NO FAX NO E MAIL EQUIPMENT Model Serial No If on loan evaluation Start Date Finish Date Hazardous materials used with this equipment Has the equipment been cleaned and decontaminated YES NO delete Method of cleaning decontamination NAME POSITION HEAD OF DIV DEP INSTITUTE COMPANY SIGNED DATE PLEASE RETURN THIS FORM TO W
11. Whatman CELL PORATOR E coli Electroporation System Order No 11613015 USA Instruction Manual Table of Contents 1 Notices to Customer i 5s ycn k dat 1 1 1 2 WGI oeste ld 1 2 OvetviewW edente tape in deste bb e edle to sr EROR t 2 2 1 DeSeri m 2 2 2 COMPONEN ses ieri 2 2 3 Introduction to 2 5 2 3 1 Background Information cde 5 2 32 Electrical Principles un ect e depre 6 2 3 3 Electroporation of E coli 2 2 0000 7 3 Operating Instructions 8 3 1 Procedural Overview EE 9 8 3 2 Setting Up the WorkAredi secca e secte ctor pan Vaca e FU Rea ana 8 3 3 Setup for Temperature Control sss 8 3 4 5 sn nene nh UR eg 9 3 5 2 02020 000020 00 10 4 Troubleshooting Guide 222 2 9 11 5 Ap liGolio AAA serien 13 5 1 Preparing E coli Cells for 2 2 0 13 5 2 Preparing DNA for Electroporation into 14 5 2 1 Preparing DNA from Ligation Reactions
12. as described in Chapter 3 Applications 5 2 Preparing DNA for Electroporation into E coli Taking extra care in the preparation of DNA for high voltage electroporation into E coli will improve both transformation efficiency and consistency DNA used for transfor mation must be free of phenol ethanol and detergents In addition electroporation is more efficient if the DNA is in a buffer of very low conductivity such as 0 5x TE If DNA is not in a low conductivity buffer e g 1 mM NaCl it should be precipitated with ethanol and resuspended in 0 5 TE buffer prior to transformation as described below It is important to remember that very small volumes of the ethanol buffer mix have a high concentration of ions Remove the ions present in the remaining ethanol buffer solution by washing with 7096 ethanol When working with small quantities of DNA it may be preferable to remove as much of the ethanol buffer mix as possible recentrifuge the tube briefly and then carefully remove all of the remaining ethanol mix with a micropipette Transfer RNA 5 to 10 ug can be used as a carrier 5 2 1 Preparing DNA from Ligation Reactions Techniques for precipitating DNA are described in references 27 and 28 1 To a 20 ul ligation reaction add 2 ul of 3 M sodium acetate pH 7 0 and mix Add 44 ul of 95 or 100 ethanol and mix 2 Centrifuge for 15 min at room temperature or at 4 at the maximum speed of the microcentrifuge e g 12 00
13. by exposing cells to a brief electrical pulse or pulses of high field strength This technique requires suspending cells between two electrodes to which a controlled unidirectional electrical pulse is applied from a power supply see figure 4 Power supply pulse generator Ec ni 9 Pa AJ Electrodes Cell suspension Figure 4 Basic Design of an Electroporation System The electroporation technique originated from studies of electric field effects on biopoly mers and organelle membranes In these studies homogeneous electric field pulses induced or enhanced material exchange through membranes 1 3 and also caused membranes to fuse when in close contact with each other a phenomenon termed elec tro cell fusion 4 5 The first successful transfer of DNA into mammalian cells by electroporation was de scribed in 1982 6 Since then this method has been used successfully for introducing foreign DNA into mammalian plant and microbial cells 7 14 More recently other high and low molecular weight molecules such as RNA 15 proteins and dyes 16 and nucleotides 17 have been introduced into cells by this method The molecular mechanism of electroporation is not well understood lt is assumed that an electric field induces a potential difference across the membranes of the cells exert ing pressure on and subsequently thinning the membrane When the induced potential difference is increase
14. ction After equilibrating to 4 C the base will remain stable at 4 C for 30 min at room temperature 1 Remove the single safe lid from the base by turning the threaded outer ring counter clockwise and lifting the lid away 2 Unscrew the two halves of the base Invert the upper half of the base to reveal the temperature control compartment 3 Fill the temperature control compartment with 20 ml of water 4 Thread the lower half of the base snugly back onto the upper half Return the reassembled base to the upright position If any leaking occurs at the joint between the two halves twist them to tighten the seal If leaking persists please call the Whatman Service Department or Technical Support 5 the base separately under refrigeration or on ice to bring to 4 Note Do not place the base at 0 or less Expansion of freezing water can cause irreparable damage to the base 6 Periodically check the amount of fluid in the temperature control compartment and refill as necessary to ensure optimal performance 3 4 Setup for Electroporation Before performing the following steps review Figures 1 through 3 as needed 1 Place the E coli pulser AC power cord and single safe lid on a flat uncluttered workspace at room temperature away from the risk of splashing or spilling water Note The main POWER switch on the E coli pulser should be in the off O posi tion Co
15. d beyond a critical point localized breakdown of the membrane formation is thought to occur If the field strength and duration of the electric pulse is not too great the pores close again and the cell survives excessive field strength or pulse length however can irreversibly damage the membrane and lyse the cells Overview 2 3 2 Electrical Principles Two methods can be used to apply the pulse for electroporating cells The output of a power supply can be used directly applied across the sample for a length of time deter mined by an electronic or mechanical switch Alternatively the output of the power sup ply can be used to charge a capacitor an electrical reservoir to a selected initial volt age which is then released as a pulse of electrical current through the sample this is the method used with the CELL PORATOR E coli System Note The two pulsing methods make different demands on power supplies and may require different experimental conditions for optimal results The most important electrical parameters in an electroporation experiment are field strength and pulse length Field strength expressed in volts per centimeter V cm is defined as the voltage applied by the power supply to the electrodes divided by the dis tance between the electrodes The field strength is determined by the design of the power supply and the shape and dimensions of the sample chamber Pulse length is determined by the c
16. d into the VAC INPUT connector on the rear control panel and into a suitable wall outlet The fuse in the VAC INPUT connector may have tripped Remove and replace it as necessary The AC outlet circuit may be tripped Check it with other equipment The POWER indicator light bulb is burned out Contact the Whatman Service Department or Technical Support Contact the Whatman Service Department or Technical Support The POWER indicator light bulb is burned out Verify that the READY indicator light is on before pressing the TRIGGER button Contact the Whatman METE Service Department or Technical Support Arcing occurred in the electroporation chamber Turn off and unplug the E coli pulser Disconnect the single safe and examine it for damage Remove and discard the chamber If the problem recurs sample conductivity may be too high see comments in Procedure troubleshooting Check that the upper and lower halves of the single safe base are screwed together snugly Tighten by turning against each other Refill the temperature compartment if necessary Examine the O ring between the two halves of the base If it appears damaged or degraded or if it does not sit properly in its groove in the lower half of the base call for a replacement If leaking persists call the Whatman METETE Service Department or Technical Support Problem Procedure When the chamber is opened th
17. e Microelectroporation N Chamber Figure 6 Schematic Diagram of CELL PORATOR E coli System Pulse Circuit 2 3 3 Electroporation of E coli Parameters Efficient electroporation of E coli 18 21 requires high sample density 1 x 1011 cells ml and field strengths as great as 20 000 V cm Transformation efficiencies achieved with this process may be 10 times greater than with cells rendered competent by chemical methods 22 Using the plasmid pUC19 and certain E coli strains that have been rendered electrotransformable efficiencies of gt 1 x 1010 transformants ug pUC19 DNA may be obtained 18 In fact such efficiencies are routine with derivatives of the F coli strain MC1061 23 24 Another advantage of the electrotransformation procedure is that a single reaction can generate over 10 transformants from as little as 50 ng of input plasmid DNA 18 These two factors high efficiency and high saturation capacity make electroporation the method of choice for the construction of large cDNA libraries from limiting input DNA or any other application in which DNA is the limiting factor such as plasmid rescue 24 construction of genomic banks 25 or working with methylated cDNA 26 In electroporation of high resistance samples such as electrotransformable cells pulse lengths v are approximately proportional to the value of the internal shunt resistor The capacitive decay shape of a pulse generated by the E coli pulser
18. e sample is splattered over the inside of the chamber Cells run down inside of chamber instead of forming a bead between the bosses Cells are slightly foamy after being pulsed Cells are obviously lysed after being pulsed No transformants result when cells are pulsed with a known DNA Transformation efficiency is less than expected or some other symptom of incorrect voltage setting Comment Sample conductivity is too high Reduce the ionic strength of the sample see Section 5 2 Use a micropipette with a delivery system that allows you to easily control the rate that the sample is applied between the bosses Apply the sample slowly first making contact with one of the bosses and then drawing the sample out to the other boss Do not try to electroporate more than 20 ul at one time This condition is normal in electroporation of E coli However to verify that the field strength is not too high reduce the VOLTAGE RANGE setting and compare the cell viability and transformation obtained The field strength was substantially too high Reduce the VOLTAGE RANGE setting Reduce the conductivity of the sample Do not attempt to electroporate high conductivity samples gt 1 mM NaCl with this system Verify that all electrical connections are correct Verify that the outer ring on the single safe lid is not being cross threaded resulting in poor electrical contact All components should be snug when the ring is properly tight
19. ened Test a batch of cells of known efficiency If VOLTAGE RANGE selector switch is set to HIGH or LOW reset to MEDIUM and repeat electroporation If VOLTAGE RANGE selector switch is set to MEDIUM compare results with those obtained using the HIGH and LOW settings 12 Applications 5 1 Preparing E coli Cells for Electroporation E coli cells suitable for high efficiency transformation by electroporation can be prepared from log phase cultures by washing the cells twice and resuspending them at a high cell density 1 x 1011 cells ml in 1096 glycerol The transformation efficiency of different strains will vary greatly In our tests the protocol that follows yields 1 x 1010 transformants ug pUC19 DNA with a number of strains table Table Expected Electrotransformation Efficiencies of E coli Strains with pUC19 DNA Strain Efficiency transformants Lg e HB101 1 0 to 1 8 x 1010 LE392 1 510 3 8 x 1010 K802 1 3 to 2 4 x 1010 JM109 0 5 to 1 1 x 1010 MC1061 4 0 to 7 0 x 1010 5 0 5 to 1 6 x 1010 All values are based on a sample size of 20 ul and a field strength of 16 6 kV cm 1 Use a fresh colony of cells to inoculate 50 ml of S O B medium without magne sium see Section 5 3 in a 500 ml flask Grow cells with vigorous aeration overnight at 37 2 Dilute 0 5 ml of cells into 500 ml of S O B medium without magnesium in a 2 8 L flask Grow for 2 to with vigorous aerat
20. exceeds 1 x 10 cells ml although transforma tion efficiency is still efficient at 1 x1010 cells ml 10 transformants ug pUC19 DNA Cell Physiology Cell physiology can also have a great impact on transformation efficiency In initial experiments E coli cells should be grown in a rich medium and harvested early in the logarithmic phase optimal conditions may then be deter mined by growing the cells in different media and harvesting at different stages of growth Temperature The transformation efficiency of electroporation of E coli is optimal when the temperature is 4 Cells DNA and disposable microelectroporation chambers should be kept on ice The single safe base with its temperature control compartment filled with 20 ml of water should be equilibrated to 4 C prior to elec troporation 14 5 3 Other Factors Transformation efficiency is reduced when gt ul of 0 5x TE buffer is added to 20 of cells formulations for 0 5x and 50x TE buffers are listed in Section 5 3 Transformation frequency is reduced at least 10 fold when gt 1 ul of ligation buffer is added to 20 ul of cells see Section 5 2 1 After electroporation cells should be diluted into 1 ml of S O C medium see Section 5 3 The cells should then be shaken for 1 h at 37 C to allow expres sion of the transformed phenotype before being plated on selective medium Medium and Buffer Formulations S O B Medium without magnes
21. f E coli at a voltage of 2 500 VDC using dis posable microelectroporation chambers The internal power supply is capable of gener ating voltages up to 3 000 VDC between the raised bosses of disposable microelectro poration chambers field strengths up to 20 000 V cm may be generated The POWER switch on the front control panel see figure 2 connects the F coli Pulser to the line current the POWER indicator light illuminates when the POWER switch is set to ON The three position VOLTAGE RANGE selector switch may be used to vary the volt age output between LOW 1 5 kV MEDIUM 2 5 kV the optimized setting for E coli cells and HIGH 2 9 kV The CHARGE button is then used to charge the capaci tor to the voltage determined by the VOLTAGE RANGE selector switch Once the unit is charged to the desired voltage the READY indicator light will come on and the LCD voltmeter will display the charge voltage The TRIGGER button is used to deliver the desired voltage to the single safe When the TRIGGER button is pushed a tone sounds to indicate that the voltage is delivered CELL PORATOR E coli Pulser O o O VOLTAGE kV CHARGE TRIGGER H MEDIUM H VOLTAGE RANGE Whatman Figure 2 E coli Pulser Front Control Panel Electroporation occurs within the single safe which contains a slot for one di
22. hatman OR YOUR LOCAL BIOMETRA DISTRIBUTOR TOGETHER WITH THE EQUIPMENT PLEASE ATTACH THIS CERTIFICATE OUTSIDE THE PACKAGING INSTRUMENTS WITHOUT THIS CERTIFI CATE ATTACHED WILL BE RETURNED TO SENDER Additional Information 8 6 Warranty Whatman MENTA warrants apparatus of its manufacture against defects in materials and workmanship under normal service for one year from the date of receipt by the pur chaser This warranty excludes damages resulting from shipping misuse carelessness or neglect Whatman s liability under the warranty is limited to the repair of such defects or the replacement of the product at its option and is subject to receipt of reasonable proof by the customer that the defect is embraced within the terms of the war ranty All claims made under this warranty must be presented to Whatman within one year following the date of delivery of the product to the customer This warranty is in lieu of any other warranties or guarantees expressed or implied aris ing by law or otherwise Whatman IEEE makes no other warranty expressed implied including warranties of merchantability or fitness for a particular purpose Under no circumstances shall Whatman NETA be liable for damages either consequen tial compensatory incidental or special sounding in negligence strict liability breach of warranty or any other theory arising out of the use of the product listed herein Whatman EEES reserves the
23. ion at 37 C until ODssq 0 8 3 Harvest cells by centrifuging at 5 000 rpm 2 600 x 9 in a GSA or GS3 rotor for 10 min 4 Wash the cell pellet by resuspending in 500 ml of sterilized ice cold WB 1096 Ultra Pure glycerol 9096 distilled water v v Centrifuge the cell suspension at 5 000 rpm 2 600 x g for 15 min and carefully pour off the supernatant as soon as the rotor stops Note Cells washed in WB do not pellet well If the supernatant is turbid increase the centrifugation time 5 Repeat step 4 in its entirety 6 Resuspend the cell pellet in WB to a final density of 200 to 250 ODs55g units 1 to 2 ml Usually no additional WB needs to be added to the cell pellet it can be resuspended in the WB that remains in the centrifuge bottle Cells can be used immediately or can be frozen in 200 ul aliquots in microcentrifuge tubes using a dry ice ethanol bath Store frozen cells at 70 Note A greater volume of lower efficiency cells can be obtained by resuspending the cells in WB to 10 ml instead of 2 ml These cells which typically will yield 5 to 10 fold fewer transformants Ug may be frozen 0 5 ml aliquots 7 Aliquot 20 ul samples of cells not frozen into sterilized microcentrifuge tubes Add 1 ul of DNA see Section 5 2 1 and mix with the pipette tip 8 Use a micropipette to suspend each cell DNA sample between the electrode boss es of a disposable microelectroporation chamber and perform electroporation
24. ium Component Amount Bacto tryptone 20g Bacto yeast extract 5g NaCl 0 584 g KCl 0 186 g distilled water toll Mix components adjust pH to 7 0 with NaOH and autoclave 2 Mgt stock Component Amount MgCl 6H5O 20 33 g MgSO 7H20 24 65 distilled water to 100 ml Autoclave or filter sterilize 2M glucose Component Amount glucose 36 04 g distilled water to 100 ml S O C Medium Filter sterilize Add 1 ml of 2 M Mg stock and 1 ml of 2 M glucose to 98 ml of S O B medium 50x TE Buffer Component Amount Tris base 12 11 9 Na EDTA 3 729 Dissolve Tris and EDTA 100 ml of water Titrate with HCI to pH 7 4 Bring volume to 200 ml with distilled water Filter sterilize or autoclave For 0 5x TE buffer add 1 ml of 50x TE buffer to 99 ml of distilled water and filter sterilize References ms 25 26 27 28 Neumann E and Katachalsky 1972 Proc Natl Acad Sci USA 69 993 Neumann E and Rosenheck K 1972 J Membr Biol 14 194 Zimmermann U 1982 Biochim Biophys Acta 694 227 Scheurich P Zimmermann U Mischel M and Lamprecht 1980 Z Naturforsch Teil C Biochem Biophys Biol Virol 37 1081 Neumann Gerisch G and Opatz 1980 Naturwissenschaften 67 414 Neumann E Schaefer Ridder M Wang Y and Hofschneider P H 1982 EMBO J 1 841 Potter H Weir L and Leder P 1984 Proc Natl Acad Sci USA 81 7161 Smithies O
25. kV 0 15 and of achieving transformation efficiencies of gt 1 x 1010 trans formants ug pUC19 DNA This manual provides instructions for proper setup and opera tion of the CELL PORATOR E coli Electroporation System and its components along with protocols for the preparation and electroporation of electrotransformable coli strains 2 2 Components The CeLL PORATOR E coli Electroporation System is designed to function as a complete integral system providing the highest possible degree of safety and convenience for the electroporation of F coli cells Each system component is engineered for durability and ease of use Refer to Figure 1 to identify the following features and components coli Pulser Single Safe with attached pulse cable Disposable Microelectroporation Chambers not shown Instruction Manual not shown Many of these components are also available separately For ordering information refer to Chapter 7 Related Products E coli Pulser Single Safe with attached pulse cable WE Figure 1 CELL PORATOR E coli Electroporation System Note Do nof freeze the base assem bly Expansion of freezing water can cause irreparable damage to the base unit The E coli Pulser delivers electrical pulses to bacterial cells by capacitor discharge The unit is equipped with a 2 fixed capacitor and a 4 internal shunt resistor that pro vide optimal electroporation efficiencies o
26. nal cautions Do not submerge the apparatus in any liquid Do not allow any electrical parts to come into direct contact with liquid Do not autoclave or dry heat sterilize the apparatus Do not expose the apparatus to phenol acetone benzene halogenated hydrocar bon solvents undiluted laboratory alcohols or any corrosive chemicals Avoid prolonged exposure of the apparatus to UV light Do not expose the apparatus to heat moisture or excessive dust Important Each disposable microelectroporation chamber should be used only once for reliable and consistent results and should be disposed of properly following use Auto claving distorts the chambers and does not remove or destroy residual DNA or other material in the crevices between chamber walls and electrodes Agents such as bleach that destroy DNA and other organic compounds will damage the aluminum electrodes and alter their electrical properties 8 2 Maintenance The following simple routine inspection and maintenance procedures will help ensure both the safety and the performance of the CeLL PORATOR E coli System 1 Inspect all electrical connections regularly If the connectors to either the pulse control or the chamber safe do not appear to be in proper working condition replace the pulse cable or call the Whatman IIEITTETZ Service Department or Technical Support 2 Ifthe single safe lid or pulse cable shows any signs of wear or damage e g fray ing c
27. nnect the pulse cable on the single safe lid to the coaxial PULSE OUTPUT con nector on the rear control panel of the E coli pulser The connection is made by inserting the end of the cable into the connector and twisting to lock in place Plug the AC power cord into the 115 VAC or 220 240 VAC depending on the specifications of your unit INPUT receptacle on the rear control panel of the E coli pulser Note Check that the fuse in the AC INPUT connector is installed and not visibly damaged Verify that the main POWER switch on the E coli pulser is set to the off O posi tion Connect the AC power cord to a suitable grounded 115 V or 220 240 V depending on the specifications of your unit power outlet Set the VOLTAGE RANGE selector switch to the MEDIUM setting Recheck all electrical connections Turn the POWER switch on the E coli pulser to the on I position Note The POWER indicator light on the front panel will light up to indicate that the unit is connected to the line current Remove the single safe base from cold storage and place it in front of the E coli pulser with the positioning pole closest to you Note This orientation facilitates operation by allowing the pulse cable on the assem bled single safe to extend straight back to the E coli pulser To test out the equipment functions begin by orienting the single safe lid over the base using the positioning post and tightening the threaded outer ring to secure
28. ombination of capacitance expressed in farads F and resistance expressed in ohms Q in a circuit Capacitance depends on the incorporation of capacitors in the circuit resistance is largely determined by the sample In the capacitor charge pulsing method the voltage of the pulse starts at the initial value and decays exponentially as a function of time according to the following equation V t Voe RC 1 where Vo is the voltage at time zero e is a constant 2 72 R is the resistance and is the capacitance F A plot of this function is shown in figure 5 Voltage et Vo 1 2 RC 3 RC Time Pulse Length Figure 5 Capacitor Discharge Curve The pulse length of a capacitor discharge is defined as the time required for the voltage to drop to 1 e 0 37 of its original value The pulse length can be calculated from equation 1 t RC 2 where is expressed in seconds 5 R is expressed Q and C is expressed In the CELL PORATOR E coli pulser an internal DC power supply charges 2 UF capaci tor to the desired voltage and the stored energy is then discharged as a controlled elec trical pulse through the disposable microelectroporation chamber and a parallel internal resistor A simplified diagram of this control circuit is shown in Figure 6 Capacitor Internal Resistor DC Power Supply 752 Disposabl
29. racking nicks abrasions or melted insulation replace it immediately 3 Do not open the instrument case of any F coli pulser These apparatus contain no user serviceable parts If you suspect internal problems with any component of this system call the Whatman EENES Service Department or Technical Sup port immediately 18 8 3 Specifications E coli Pulser 3 4 kg 7 5 Ib Dimensions WX EXHI a 23 6 x 26 6 x 14 0 cm 9 3 x 8 75 x 5 5 in Case mote tana ada eeiam dese be KEN aluminum steel Power requirements Cat Nov 116132049 se en nenn 100 115 VAC 50 60 Hz 60 W SOP OSI CC ee 2 uF Internal rice 4 Q parallel to chamber Pulse output receptacle SHV Receptacle Kings 1704 1 Maximum pulse current AWA K di 40A Digital LCD 5 accuracy 5 20 Single Safe 1 2 kg 2 6 Ib Dimensions sarah 7 0x 12 1 cm 2 75 x 4 75 in Temperature control compartment volume E 20 ml Con Sir c stainless steel DELRIN A eds attached SHV Plug Kings 1705 2 30 long coaxial cable Disposable Microelectroporation Chamber Ove
30. rall dimensio N Sisus 4 3 x 4 8 x 3 0 cm W x Lx H 1 7 x 1 9 x 1 2 in Construction polypropylene aluminum Working pev deo b yo 20 ul Average electrode dap ite r mm n Ped Re enia 0 15 cm Additional Information 8 4 Service Should you have any problems with this unit please contact our service department the technical support or your local Biometra dealer Whatman METAS Service Department Rudolf Wissell StraBe 14 16 D 37079 G ttingen Germany Phone 49 0 5 51 50 88 1 10 or 12 Fax 49 0 5 51 50 88 1 11 Mail service biometra co uk Whatman HIE Technical Support 9101 Yellow Brick Road Suite A Baltimore MD 21237 USA Phone 866 793 7671 toll free Fax 410 574 2380 Instructions for return shipment IMPORTANT Before sending the unit back to us it is urgently necessary also to call our service depart ment the technical support or your local Biometra dealer Return only defective devices For technical problems which are not definitively recognis able as device faults please contact the Whatman EEEE Service Department or Technical Support Use the original box or a similarly sturdy one Label the outside of the box with CAUTION SENSITIVE INSTRUMENT Please enclose a precise description of the fault which also reveals during which proce dures the fault occurred if possible Important
31. right to make improvements in design construction and appearance without notice 8 7 Declaration of Conformity and CE Mark Note The information outlined in this section applies only to customers located in the European Union EU The EU is currently comprised of 15 member countries This laboratory apparatus is identified with the CE mark This mark indicates that the product complies to the following EU Directives and Standards Application of Council Directive s 73 23 EEC Low Voltage Directive 89 336 EEC Electromagnetic Compatibility Standards EN 61010 1 1993 Product Safety EN 50081 1 1992 Emissions EN 50082 1 1992 Immunity Address Whatman BETETEN Rudolf Wissell Str 30 37079 G ttingen Germany phone 49 0 551 50686 0 fax 49 0 551 50686 66 A copy of the Declaration of Conformity certificate is available upon request 22 Notes 23 24 Notes PartNo 14332 Lot No 01 0200
32. sposable microelectroporation chamber see figure 3 The lid of the single safe includes a coaxial pulse cable that connects to the high voltage PULSE OUTPUT connector on the E coli Pulser The base of the single safe is a thermal mass that may be used to help maintain the desired sample temperature of 4 for electroporation Unscrewing the base reveals a temperature control compartment that may be filled with 20 ml of water The entire base assembly may be kept separately under constant refrigeration at 4 or on and removed to the work area for electroporation After each disposable microelectropora tion chamber is loaded into the base the lid is oriented for electroporation using the positioning post and electrode rivets The threaded outer ring that secures the lid in place for electroporation is then tightened Overview Radiation sterilized individually packaged disposable microelectroporation chambers see figure 3 are designed for high transformation efficiencies of E coli and other bacte rial samples These chambers are made of translucent polypropylene and have leak proof caps to allow mixing of the cell suspension Each of these chambers contains two integral flat aluminum electrodes separated by an average distance of 0 15 cm The two rivets through the of each chamber make contact with both the electrodes and the spring loaded contacts of the single safe lid when the lid is closed Positioning post
33. stered trademark of E l du Pont de Nemours amp Co DH5a and Focus are marks of Invitrogen Corporation Notices to Customer 1 1 Important Information Read and carefully follow manual instructions Do not alter equipment Failure to adhere to these directions could result in personal and or laboratory hazards as well as invalidate equipment warranty This product is authorized for laboratory research use only The product has not been qualified or found safe and effective for any human or animal diagnostic or therapeutic application Uses for other than the labeled intended use may be a violation of applica ble law 1 2 Warnings 1 DANGER Although designed to prevent accidental exposure to high voltages this apparatus should always be operated with extreme caution Do not attempt to defeat safety interlock features designed for user protection Careless handling could result in a lethal electric shock This system should be operated by trained personnel only This manual should be readily accessible to all users 2 Never operate damaged or leaking equipment 3 Use only disposable microelectroporation chambers with the CELL PORATOR E coli System 4 not attempt to electroporate high conductivity samples gt 1 mM NaCl with this system 5 Before operating this system make sure that all electrical connections are secure and that power cords show no signs of damage 6 Avoid spilling liquid on or into the apparat
34. the lid in place Note Do not load a disposable microelectroporation chamber into the single safe at this time Press the CHARGE button to charge the capacitor Note When the unit is ready to deliver a pulse the READY indicator light on the front panel will be lit With the VOLTAGE RANGE selector switch set to MEDIUM the LCD voltmeter should display a reading of 2 5 kV Confirm that the READY indicator light is lit and push the TRIGGER button to deliver the pulse Note tone sounds to indicate that the voltage is delivered and the LCD voltmeter reading should drop to 0 Repeat steps 7 and 8 once with the VOLTAGE RANGE selector switch set to LOW and again with the switch set to HIGH The only change from the first trial run should be the LCD voltmeter readout prior to delivering the pulse 1 5 kV at the LOW set ting and 2 9 kV at the HIGH setting Check the voltmeter to make sure the capacitor is discharged the LCD voltmeter should read 0 and turn the POWER switch on the E coli pulser to the off O position Disconnect the E coli pulser from the AC outlet Remove the single safe lid from the base and return the base to cold storage 4 until needed Operating Instructions 3 5 2 9 o Electroporation Mark the required number of culture tubes and add 1 ml of S O C medium to each Keep these tubes to one side at room temperature for use later in this proto
35. us Do not submerge the apparatus in any liquid Do not operate the apparatus if it is accidentally wetted 7 Be sure that the E coli Pulser is discharged and turned off before connecting or dis connecting the pulse cable 8 Do not open the instrument case of any CELL PORATOR E coli System component These apparatus contain no user serviceable parts 9 Always connect the CELL PORATOR E coli System to a 3 wire grounded AC power outlet Do not alter the grounded AC power cord provided with the unit Do not use a two wire receptacle with an adapter This could create a serious electrical hazard for persons using this unit 10 For maximum safety always operate this system in an isolated low traffic area that is not accessible to unauthorized personnel 11 Certain reagents indicated for use in this manual are of a hazardous nature researcher is cautioned to exercise care with these reagents and with the equipment used in these procedures following the manufacturers safety recommendations 12 This system is designed for use at room temperature Do not set up for operation in a cold room or other low temperature environment Overview 2 1 Description The CeLL PORATOR E coli Electroporation System is an electroporation device designed to introduce DNA into Escherichia coli with optimal efficiency reproducibility safety and convenience This system is capable of generating field strengths as high as 20 000 V cm 3 0
Download Pdf Manuals
Related Search