DR Field Bedienerhandbuch

CHEF-DR®III Pulsed FieldElectrophoresis SystemsInstruction Manualand Applications GuideCatalog Numbers 170-3690through170-3703For Technical Service Ca

If the system includes the Cooling Module, connect the temperature probe cable to theREMOTE SENSOR port on the rear panel of the Cooling Module. Inser

Block Program from 1–3 Blocks. Block 1 is run first, then Block 2, thenBlock 3. A run time of 0 disables a Block.Initial Switch Time Adjust from 0.1–6

Program TerminationThe program in progress may be manually terminated by holding down PAUSE/START RUNfor 3– 4 seconds. A program can be terminated onl

3.2 Liquid SamplesHigh molecular weight DNA can be prepared by standard procedures. DNA fragments ofup to several hundred kilobases do not require pre

3.4 Preparation of Agarose Embedded Bacterial DNAThe buffers, enzymes, and agarose in the following procedure are provided in the CHEFBacterial Genomi

8. Remove the lysozyme buffer and rinse the plugs with 25 ml of 1x wash buffer (see step9 for wash buffer recipe). Add 5 ml of Proteinase K Reaction B

8. Using a 50 ml conical centrifuge tube, add 5 ml of lyticase buffer (10 mM Tris, pH 7.2, 50mM EDTA, 1 mg/ml lyticase) for each 1 ml of plugs. Push t

Fig. 3.1. Hemocytometer grid.Mammalian or tissue culture cellsBecause of the large size, tissue culture cells can be counted at 100x power. Count 10 o

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=

Bacterial:(4.5 x 106bp)(660 g/mole) = 4.933 x 10-15g DNA/cell (A)6.02 x 1023bp/mole(4.933 x 10-15g DNA/cell)(5 x 108cell/ml) = (B)(2.467 x 10-6g/DNA

WarrantyThe CHEF-DR III power module, chamber, variable speed pump, and accessories arewarranted against defects in materials and workmanship for 1 ye

173. To attach the desired comb to the comb holder, place the comb over the 2 metal pins, andturn the screw clockwise. This causes one pin to move tow

Before beginning the electrophoresis run, check the current output displayed on the CHEF-DR III power module to insure that the correct buffer concent

19Fig. 4.2. Lambda ladder was separated on a 1.0% Molecular Biology Certified Agarose (catalog num-ber 162-0133) gel in 0.5x TBE, recirculated at 14 °

4.7 Removing and Staining the GelBefore removing the gel, make sure the run is completed. The unit will display End. Tostain the gel during a run, pus

Fig. 5.1. Two gels, one in 0.5x TBE and the other in 1.0x TAE, were run to show the difference inmobility of DNA in the two buffers.S. cerevisiae was

Figure 5.2. Separation of S. cerevisiae chromosomes using angles from 120° to 94°.Electrophoresis Run TimeThe electrophoresis run time is determined b

5.4 Blotting Megabase DNAs†Southern Blot TransferPulsed field electrophoresis is a powerful technique for physical mapping of genes invarious organism

8. Dry the membrane by blotting onto 3MM or other adsorbent paper and proceed withhybridization. UV crosslinking of the DNA to the membrane is not rec

5.5 Separations of DNA Size Standards1. Restriction fragmentsSize Range: 0.2–23 kbAgarose: 1.2% Molecular Biology CertifiedBuffer: 0.5x TBETemperature

265. Hansenula wingeiSize Range: 1–3.1 mbAgarose: 0.8% Molecular Biology CertifiedBuffer: 1.0x TAETemperature: 14 °CSwitch Time: 500 secondsRun Time:

Table of ContentsPageSection 1 General Information ...11.1 Safety..

Section 6 Maintenance of Equipment6.1 Replacing ElectrodesThe gel chamber requires little maintenance except rinsing after every run. Dirt and otherbu

Section 7 Troubleshooting GuideProblem SolutionEquipmentNo power 1. Check fuse at back of power module2. Check source of A/C power3. Contact Bio-Rad L

Problem SolutionLarge DNAs not resolved 1. Lower the voltage to below 2 V/cm2. Increase switch time or use switch time ramp3. Agarose impuritiesHigh b

Section 8References8.1 Applications in Pulsed Field ElectrophoresisThe following references in pulsed field electrophoresis are primarily from 1987-19

Application Reference NumbersCircular DNA 8, 75, 78, 79, 104, 118, 159, 160, 163, 164, 190, 207Cosmid mapping 14, 46, 70, 98, 173, 193Diagnostics (e.g

8.2 Reference List for Pulsed Field Electrophoresis 1. Abel, K. J. and Gross, K. W., Nucl. Acids Res., 16, 2111-2126 (1988). 2. Adams, R. A., Nash, T.

41. Coulson, A., Waterson, R., Kiff, J., Sulston, J. and Kohara, Y., Nature, 335, 184-186 (1988). 42. Cowman, A. F., Morry, M. J., Biggs, B. A., Cross

77. Hennekes, H. and Kuhn, S., Anal. Biochem., 183, 80-83 (1989). 78. Hightower, R., Metge, D. W. and Santi, D. V., Nucl. Acids Res., 15, 8387-8398 (1

111. Magee, B. B. and Magee, P. T., J. Gen. Microbiol., 133, 425-430 (1987). 112. Magee, P. T., Rikkerink, E. H. and Magee, B. B., Anal. Biochem., 175

147. 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). 1

Section 1 General Information1.1 Safety The CHEF-DR III system uses high voltage and current, and should be operated withcare at all times. The safe

182. Thiele, D. J., Molec. and Cell. Biol., 8, 2745-2752 (1988). 183. Traver, C. N., Klapholz, S., Hyman, R.W. and Davis, R. W., Proc. Natl. Acad. Sci

Section 9Systems, Accessories, and Reagents for PulsedField ElectrophoresisCatalogNumber Product Description170-3700 CHEF-DR III Chiller System, 120 V

CatalogNumber Product Description170-4322 20 Well Comb, 14 cm wide, 1.5 mm thick170-4344 30 Well Comb, 14 cm wide, 1.5 mm thick170-3623 Preparative Co

CatalogNumber Product Description170-3590 Gene-Lite Chemiluminescent Detection Kit170-3742 Standard Documentation System, 120 VAC, includes Mini-Trans

Life ScienceGroupBio-Rad Laboratories Main Office 2000 Alfred Nobel Drive, Hercules, California 94547, Ph. (510) 741-1000, Fx. (510)741-5800Also in: A

tials are readjusted immediately to maintain uniform fields, thus insuring high resolution. InPACE, the voltage potential of each of the 24 electrodes

Accessories included:Variable speed pump 120 V, ground isolated. Flow rate 1 liter/min, typi-calCasting stand 14 cm x 13 cmComb 10 well comb and comb

Figure 1.1. Voltage clamping by the CHEF-DR III system. A. Relative electrode potentials when the + 60° field vector is activated. B. Relative electro

Cooling ModuleThe Cooling Module is a stand alone, portable refrigerated apparatus specifically for usewith the CHEF-DR III system. The variable speed

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