CHEF-DR® III Pulsed Field Electrophoresis Systems Instruction Manual and Applications Guide Catalog Numbers 170-3690 through 170-3703 For Technical Service Call Your Local Bio-Rad Office or in the U.S.
Warranty The CHEF-DR III power module, chamber, variable speed pump, and accessories are warranted against defects in materials and workmanship for 1 year. If any defects occur in the instruments or accessories during this warranty period, Bio-Rad Laboratories will repair or replace the defective parts at its discretion without charge. The following defects, however, are specifically excluded: 1. Defects caused by improper operation. 2.
Table of Contents Page Section 1 General Information .....................................................................................1 1.1 1.2 1.3 1.4 Safety............................................................................................................................1 Overview......................................................................................................................1 Specifications .............................................................................
Section 1 General Information 1.1 Safety The CHEF-DR III system uses high voltage and current, and should be operated with care at all times. The safety interlocks are for your protection and should not be circumvented. To avoid shock, set up the CHEF-DR III components in a dry area. Immediately wipe up any spilled buffers or salt solutions. When pausing or aborting a run, always check that the current display goes to zero or OFF. This can take 2–5 seconds while the power supply discharges.
tials are readjusted immediately to maintain uniform fields, thus insuring high resolution. In PACE, the voltage potential of each of the 24 electrodes is regulated independently. Unlike the CHEF-DR II system, which has a fixed reorientation (field) angle of 120° due the hexagonal geometry of the electrode array, the CHEF-DR III system can generate field angles from 90–120°.
Accessories included: Variable speed pump Casting stand Comb Tygon tubing Disposable sample plug mold Yeast DNA Standard Chromosomal grade agarose Pulsed field certified agarose Leveling bubble Fuses Manual Screened cap 120 V, ground isolated. Flow rate 1 liter/min, typical 14 cm x 13 cm 10 well comb and comb holder 365 cm 50 slot S. cerevisiae YNN295, 2 plugs 5 grams 5 grams 1 0.
A. + 60° + + + B. - 60° + + – + – + – – + ➤ + + + + + + – – – + – + + ➤ + + + + + + + + + + + + + + + + + + + + + + + Figure 1.1. Voltage clamping by the CHEF-DR III system. A. Relative electrode potentials when the + 60° field vector is activated. B. Relative electrode potentials when the - 60° field vector is activated. Electrophoresis Chamber The CHEF-DR III electrophoresis cell consists of a 44.2 x 50.3cm (17.4" x 19.
Cooling Module The Cooling Module is a stand alone, portable refrigerated apparatus specifically for use with the CHEF-DR III system. The variable speed pump circulates electrophoresis buffer directly through the unique heat exchanger, which is a tube within a tube. Buffer circulates through the inner stainless steel tube, while liquid refrigerant circulates through the outer copper tube, resulting in rapid and efficient cooling at a rate of 0.75 °C/minute (from ambient temperature to 14 °C).
Variable Speed Pump Tygon Tubing Temperature Probe Cable Variable Speed Pump Cable CHEF DR III Power Module IN OUT TO INTERLOCK Cooling Module Electrophoresis Chamber 25 Pin Cable Safety Interlock Cable Output to Electrophoresis Cell Fig. 2.1. Interconnections between components of the CHEF-DR III system. Attach the power cords for the power module and Cooling Module to the back of each instrument. Be sure the power module is off.
If the system includes the Cooling Module, connect the temperature probe cable to the REMOTE SENSOR port on the rear panel of the Cooling Module. Insert the other end of the temperature probe cable into the rear of the electrophoresis chamber. Establish the correct buffer flow before attempting any electrophoresis runs. The optimal flow rate of buffer through the electrophoresis chamber is approximately 0.8–1 liter per minute (approximately 70 on the pump).
Block Program from 1–3 Blocks. Block 1 is run first, then Block 2, then Block 3. A run time of 0 disables a Block. Initial Switch Time Adjust from 0.1–65K seconds. Final Switch Time Adjust from 0.1–65K seconds. Run Time Adjust from 0.1–999 hours. A run time of 0 disables a Block. Volts/cm Adjust from 0.6–9.0 volts in 0.1 volt increments. Included Angle Adjust from 90–120° in 1° increments. Actual Current Displays the current, in mA, provided by the power supply.
Program Termination The program in progress may be manually terminated by holding down PAUSE/START RUN for 3– 4 seconds. A program can be terminated only while it is in the run mode; it can not be terminated in PAUSE. When the program is terminated two beeps will sound, and the right display will show OFF. Pressing PAUSE/START RUN again will start the program from the beginning.
3.2 Liquid Samples High molecular weight DNA can be prepared by standard procedures. DNA fragments of up to several hundred kilobases do not require preparation in agarose blocks, and can be added to the wells in liquid form. When working with DNA in the range of 50–200 kb, it may be necessary to use pipette tips with large openings. When running only liquid samples, the best resolution and sharpness of bands is achieved using a thin well comb (0.75 mm). 3.
3.4 Preparation of Agarose Embedded Bacterial DNA The buffers, enzymes, and agarose in the following procedure are provided in the CHEF Bacterial Genomic DNA Plug Kit (catalog number 170-3592; see Section 9 for information). 1. Inoculate a bacterial culture into 50 ml of LB Broth or appropriate media and grow with agitation to an O.D.600 of 0.8–1.0 at the appropriate temperature. 2. When the desired O.D.
8. Remove the lysozyme buffer and rinse the plugs with 25 ml of 1x wash buffer (see step 9 for wash buffer recipe). Add 5 ml of Proteinase K Reaction Buffer (100 mM EDTA, pH 8.0, 0.2% sodium deoxycholate, 1% sodium lauryl sarcosine, 1 mg/ml Proteinase K) for each ml of agarose plugs. Incubate the plugs overnight at 50 °C without agitation. Note: various cell lines have been incubated up to 4 days in Proteinase K without detrimental effects to the quality of DNA. 9.
8. Using a 50 ml conical centrifuge tube, add 5 ml of lyticase buffer (10 mM Tris, pH 7.2, 50 mM EDTA, 1 mg/ml lyticase) for each 1 ml of plugs. Push the solidified agarose plugs, using the snap off tool provided on the plug mold, into the 50 ml centrifuge tube containing the lyticase buffer. Incubate the plugs 30 minutes to 1 hour at 37 °C without agitation. 9. Remove the lyticase buffer and rinse the plugs with 25 ml of 1x wash buffer (see step 10 for wash buffer recipe).
B C A Fig. 3.1. Hemocytometer grid. Mammalian or tissue culture cells Because of the large size, tissue culture cells can be counted at 100x power. Count 10 of the large squares, five on each side of the hemocytometer.
For Example: 300 bacteria in 5 squares = average of 60 bacteria/square x 25 (squares) x 20 (dilution factor, yeast use 100 for dilution factor) x 104 = 3 x 108 bacteria per ml. So for 5 ml of plugs you need 5 ml x 5 x 108 cells final concentration ÷ 3 x 108 actual cells concentration = 8.33 ml of cell suspension is required. 3.8 Estimation of Agarose Plug DNA Concentration Two pieces of information are needed to determine DNA concentration: 1. The size in base pairs of the genome.
Bacterial: 6 (4.5 x 10 bp)(660 g/mole) = 4.933 x 10-15 g DNA/cell 6.02 x 1023 bp/mole (A) (4.933 x 10-15 g DNA/cell)(5 x 108 cell/ml) = (B) (2.467 x 10-6 g/DNA/ml)(1 x 106 µg/ml) = 2.5 µg DNA/ml (2.5 µg DNA/ml)(2.5 genome equivalents) ≅ 6.25 µg DNA/ml (6.25 µg DNA/ml) 10 plugs/ml ≅ (0.625 µg DNA/plug) 2 lanes/plug ≅ 0.3 µg DNA/lane (C) (D) Yeast: (1.5 x 107 bp)(660 g/mole) = 1.644 x 10-14 g DNA/cell 6.02 x 1023 bp/mole (A) (1.644 x 10-14 DNA/cell)(6 x 108 cell/ml) = (B) (9.
3. To attach the desired comb to the comb holder, place the comb over the 2 metal pins, and turn the screw clockwise. This causes one pin to move towards the screw, holding the comb in place. Adjust the height of the comb to 2 mm above the surface of the platform by loosening the screw (counterclockwise), then tightening when the comb is properly positioned. A thin plastic ruler makes a good height gauge. 4.
Before beginning the electrophoresis run, check the current output displayed on the CHEF-DR III power module to insure that the correct buffer concentration is used. The following values are for 2 liters of buffer at 14 °C circulating through the electrophoresis cell. Buffer Concentration 0.5x TBE (at 14 ˚C) 0.5x TBE (at 14 ˚C) 0.5x TBE (at 14 ˚C) 0.5x TBE (at 14 ˚C) Voltage Gradient 2 V/cm 3 V/cm 6 V/cm 9 V/cm Current Range 30–45 mA 50–65 mA 115–135 mA 190–210 mA 1.0x TAE (at 14 ˚C) 2 V/cm 75–90 mA 1.
Lambda Ladder S. cerevisiae H. wingei S. pombe Fig. 4.2. Lambda ladder was separated on a 1.0% Molecular Biology Certified Agarose (catalog number 162-0133) gel in 0.5x TBE, recirculated at 14 °C. The run time was 22 hours at 6 V/cm with a 50 to 90 second switch time ramp at an included angle of 120°. Saccharomyces cerevisiae Strain YNN295. Chromosomes were separated on a 1.0% Pulsed Field Certified Agarose (catalog number 162-0137) gel in 0.5x TBE, recirculated at 14 °C.
4.7 Removing and Staining the Gel Before removing the gel, make sure the run is completed. The unit will display End. To stain the gel during a run, push PAUSE/START RUN on the CHEF-DR III system. Remove the gel (on the platform) from the cell, slide it off the platform into a 0.5 µg/ml ethidium bromide solution in water, and let the gel stain for 20–30 minutes. (Caution: Ethidium bromide is a mutagen. Always wear gloves while handling gels or solutions containing the dye.
Size (kb) 2,400 2,200 2,000 1,800 1,600 1,400 1,200 1,000 800 600 400 200 0 0.0 1.0x TAE 0.5x TBE 0.1 0.2 0.3 0.4 Velocity (cm/hr) 0.5x TBE 1.0x TAE Fig. 5.1. Two gels, one in 0.5x TBE and the other in 1.0x TAE, were run to show the difference in mobility of DNA in the two buffers. S. cerevisiae was separated on a 1.0% Pulsed Field Certified Agarose (catalog number 162-0137) gel with a 60 second switch time for 15 hours, followed by a 90 second switch time for 9 hours, at 6 V/cm.
120° 105° 100° 96° 94° Figure 5.2. Separation of S. cerevisiae chromosomes using angles from 120° to 94°. Electrophoresis Run Time The electrophoresis run time is determined by the migration rates of the DNA molecules under investigation. The migration rates, in turn, are affected by the switch time, field strength, and field angle. As the migration rate of the DNA molecules decreases, the electrophoresis run time must increase to adequately resolve the DNA molecules of interest. 5.
5.4 Blotting Megabase DNAs † Southern Blot Transfer Pulsed field electrophoresis is a powerful technique for physical mapping of genes in various organisms. To determine the chromosomal location of a gene in a microorganism or the size of the restriction fragment containing a gene in mammalian systems, large DNA fragments separated by CHEF are transferred onto membranes and detected by Southern hybridization analysis.
8. Dry the membrane by blotting onto 3MM or other adsorbent paper and proceed with hybridization. UV crosslinking of the DNA to the membrane is not recommended with this alkaline transfer method. † Contributed by Dr. Eric Lai, University of North Carolina Discussion 1. The procedure is for gels approximately 6 mm thick. If thicker gels are used, the staining period may be prolonged to allow diffusion of EtBr into the middle of the gels.
5.5 Separations of DNA Size Standards 1. Restriction fragments Size Range: Agarose: Buffer: Temperature: Switch Time: Run Time: Angle: Voltage Gradient: 0.2–23 kb 1.2% Molecular Biology Certified 0.5x TBE 14 °C 0.1 second 3 hours 120° 9 V/cm 2. 5 kb Ladder Size Range: Agarose: Buffer: Temperature: Switch Time: Run Time: Angle: Voltage Gradient: 5–75 kb 1.0% Molecular Biology Certified 0.5x TBE 14 °C 1-6 seconds 11 hours 120° 6 V/cm 3.
5. Hansenula wingei Size Range: Agarose: Buffer: Temperature: Switch Time: Run Time: Angle: Voltage Gradient: 1–3.1 mb 0.8% Molecular Biology Certified 1.0x TAE 14 °C 500 seconds 48 hours 106° 3 V/cm 6. Schizosaccharomyces pombe Size Range: Agarose: Buffer: Temperature: Switch Time: Run Time: Angle: Voltage Gradient: 3.5–5.7 mb 0.8% Chromosomal Grade 1.0x TAE 14 °C 1,800 seconds 72 hours 106° 2 V/cm 7. Angle Ramp: S. pombe Size Range: Agarose: Buffer: Temperature: 3.5–5.7 mb 0.8% Chromosomal Grade 1.
Section 6 Maintenance of Equipment 6.1 Replacing Electrodes The gel chamber requires little maintenance except rinsing after every run. Dirt and other build-up can be removed with laboratory detergent and a fine cloth. Do not bend or break the electrodes. Fast switch times (<2 seconds) with high voltage gradients (6-9 V/cm) may lead to increased electrode failure If one of the electrodes should break, or leak at the O-ring, it may be replaced.
Section 7 Troubleshooting Guide Problem Solution Equipment No power 1. Check fuse at back of power module 2. Check source of A/C power 3. Contact Bio-Rad Laboratories No voltage across electrodes, with AC power light on 1. 2. 3. 4. 5. Gel floats away 1. Pump flow rate is too high. Adjust with Variable Speed Pump. No or low buffer flow 1. Look for kink in tubing 2. Check Cooling Module; buffer in the heat exchanger can freeze if the chiller is cooling, but the pump is not on 3.
Problem Solution Large DNAs not resolved 1. Lower the voltage to below 2 V/cm 2. Increase switch time or use switch time ramp 3. Agarose impurities High background in lanes 1. Insufficient washing of samples 2. Sample may be contaminated with RNA or other material 3. DNA concentration too high Distorted bands 1. 2. 3. 4. 5. 6. Thick bands 1. Use thinner wells 2. Load less sample Sample contains too high salt or detergent concentration Buffer breakdown. Change every 48 hours. Wells were distorted.
Section 8 References 8.1 Applications in Pulsed Field Electrophoresis The following references in pulsed field electrophoresis are primarily from 1987-1989. The list surveys a wide area of applications and organisms, but is not exhaustive. Underlined references use the CHEF-DR II pulsed field electrophoresis system.
Application Reference Numbers Circular DNA 8, 75, 78, 79, 104, 118, 159, 160, 163, 164, 190, 207 Cosmid mapping 14, 46, 70, 98, 173, 193 Diagnostics (e.g., cancer) 148 DNA over 5 megabases 20, 72, 130, 197 DNA under 200,000 bases 12, 34, 39, 77, 93 Epstein-Barr virus 75 FIGE 7, 12, 14, 15, 17, 24, 25, 33, 47, 65, 68, 77, 146, 173, 182, 187 FIGE with S.
8.2 Reference List for Pulsed Field Electrophoresis 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. Abel, K. J. and Gross, K. W., Nucl. Acids Res., 16, 2111-2126 (1988). Adams, R. A., Nash, T. E. and Wellems, T. E., Nucl. Acids Res., 16, 4555-4565 (1988). Albig, W. and Entian K-D., Gene, 73, 141-152 (1988). Amler, L.C. and Schwab, M., Molec. and Cell Biol., 9, 4903-4913 (1989). Bancroft, I. and Wolk, C.
41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. Coulson, A., Waterson, R., Kiff, J., Sulston, J. and Kohara, Y., Nature, 335, 184-186 (1988). Cowman, A. F., Morry, M. J., Biggs, B. A., Cross, G. A. and Foote, S. J., Proc. Natl. Acad. Sci. USA, 85, 9109-9113 (1988). Craig, J., Fowler, S., Skinner, J. D., Burgoyne, L.A. and McInnes, J. L., Applied and Theor. Electro., 1, 23-28 (1988). Crater, G. D., Gregg, M. C.
77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. Hennekes, H. and Kuhn, S., Anal. Biochem., 183, 80-83 (1989). Hightower, R., Metge, D. W. and Santi, D. V., Nucl. Acids Res., 15, 8387-8398 (1987). Hightower, R. and Santi, D.V., Electrophoresis, 10, 283-289 (1989). Hockett, R. D., de Villartay, J-P., Pollock, K., Poplack, D. G., Cohen, D. I. and Korsmeyer, S. J., Proc. Natl. Acad. Sci.
111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. Magee, B. B. and Magee, P. T., J. Gen. Microbiol., 133, 425-430 (1987). Magee, P. T., Rikkerink, E. H. and Magee, B. B., Anal. Biochem., 175, 361-372 (1988). Majiwa, P. A., Young, J. R., Hamers, R. and Mattyssens, G., Gene, 41, 183-192 (1986). Maniloff, J., Nucl. Acids Res., 17, 1268 (1989). Marchuk, D. and Collins, F. S., Nucl.
147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. Rubin, C. M., Carrino, J. J., Dickler, M. N., Leibowitz, D., Smith, S. D. and Westbrook, C. A., Proc. Natl. Acad Sci. USA, 85, 2795-2799 (1988). Russo, G., Isobe, M., Gatti, R., Finan, J., Batuman, O., Huebner, K., Nowell, P. C. and Croce, C. M., Proc. Natl. Acad. Sci. USA, 86, 602-606 (1989). Sanz, J. L., Marin, I., Ramirez, L.
182. 183. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 215. 216. 217. Thiele, D. J., Molec. and Cell. Biol., 8, 2745-2752 (1988). Traver, C. N., Klapholz, S., Hyman, R.W. and Davis, R. W., Proc. Natl. Acad. Sci. USA, 86, 5898-5902 (1989). Turmel, C. and Lalande, M., Nucl. Acids Res., 16, 4727 (1988). Upcraft, J. A., Boreham, P. F. and Upcraft, P., Nucl. Acids Res., 17, 3315 (1989).
Section 9 Systems, Accessories, and Reagents for Pulsed Field Electrophoresis Catalog Number Product Description 170-3700 CHEF-DR III Chiller System, 120 VAC, includes CHEF-DR III power module; electrophoresis cell; Cooling Module; variable speed pump; Tygon tubing, 12 feet; 14 cm wide x 13 cm long casting stand and frame; 10 well comb and comb holder; screened cap; 50 well disposable plug mold; leveling bubble; cables; 3/8 inch straight tubing connectors, 2; 0.5 A FB fuses, 2; S.
Catalog Number Product Description 170-4322 20 Well Comb, 14 cm wide, 1.5 mm thick 170-4344 30 Well Comb, 14 cm wide, 1.5 mm thick 170-3623 Preparative Comb, 14 cm wide, 1.5 mm thick, plus 2 outer sample wells for size standards 170-3627 15 Well Comb, 21 cm wide, 1.5 mm thick 170-3628 30 Well Comb, 21 cm wide, 1.5 mm thick 170-3645 45 Well Comb, 21 cm wide, 1.
Catalog Number Product Description 170-3590 Gene-Lite Chemiluminescent Detection Kit 170-3742 Standard Documentation System, 120 VAC, includes Mini-Transilluminator, 100 V, DS-34 camera, standard hood, deep yellow DS-34 camera filter 170-3746 Standard Documentation System, 100 VAC 170-3747 Standard Documentation System, 220/240 VAC 170-3743 Wide/Long Documentation System, 120 VAC, includes MiniTransilluminator, 100 V, DS-34 camera, wide/long hood, deep yellow DS-34 camera filter 170-3748 Wide/L
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