Home

Density Gradient Fractionation Systems User Manual

1. 1 4 Tubing and Lengths iii AA a ee a nn 2 7 Connection Hardware 2 7 Tubing and Lengths 2 11 Connection Hardware 2 11 Flow Cell Illuminated Volumes 2 12 Recommended Flow Rates 3 2 Collar and Ring Selection 3 8 Viscosity of various salt solutions used for density gradients A 2 Density at 25 C of various solutions used for density gradients A 3 Density of aqueous sucrose solutions g mL A 4 Centrifugal force chart for rotors commonly used in density gradient centrifugation A 5 Sedimentation rates and ultraviolet absorbances of some representative viruses A 6 Density Gradient Fractionation Systems Section 1 Introduction 1 1 System Features After spinning the centrifuge tubes the Density Gradient Frac tionation systems allow you to fractionate and quantitate centri fuged zones with unmatched precision The Density Gradient Fractionation systems produce a continuous UV absorbance profile as the gradient is collected in precisely measured frac tions Fractionation is performed by introducing a dense chase liquid
2. 1399 1 1391 1 1383 1 1374 1498 1 1491 1 1484 1 1477 1 1470 1 1463 1 1448 1 1440 1 1432 1 1423 1548 1 1541 1 1534 1 1527 1 1520 1 1513 1 1497 1 1439 1 1481 1 1472 1598 1 1591 1 1585 1 1577 1 1570 1 1563 1 1547 1 1539 1 1530 1 1522 0649 1 1642 1 1635 1 1628 1 1620 1 1613 1 1597 1 1589 1 1580 1 1572 1700 1 1693 1 1686 1 1679 1 1671 1 1663 1 1647 1 1639 1 1631 1 1622 1752 1 1748 1 1737 1 1730 1 1722 1 1714 1 1698 1 1690 1 1681 1 1672 j y 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1837 1 1830 1 1824 1 1817 1 1810 1 1803 1 1796 1 1789 1 1781 1 1774 1 1889 1 1883 1 1876 1 1870 1 1863 1 1856 1 1848 1 1841 1 1833 1 1825 1 1817 1801 1 1792 1 1784 1 1775 1 1 1 1 1 1766 1 1 1 1 1943 1 1936 1 1929 1 1922 1 1915 1 1908 1 0901 1 1893 1 1886 1 1878 1 1870 1853 1 1844 1 1835 1 1826 1 1 1 1 1749 1 1741 1 1732 1 1723 1 1 1 1 1 1 1 1 1 1 1996 1 1989 1 1983 1 1976 1 1968 1 1961 1 1954 1 1946 1 1938 1 1930 1 1922 1 1905 1 1896 1 1887 1 1878 1 2050 1 2043 1 2036 1 2029 1 2022 1 2014 1 2007 1 1999 1 1991 1 1983 1 1975 1 1 1958 1 1949 1 1940 1 1931 1 2104 1 2097 1 2090 1 2083 1 2076 1 2068 1 2060 1 2052 1 2044 1 2036 1 2028 1 2010 1 2002 1 1993 1 1983 1 2159 1 2152 1 2144 1 2137 1 2130 1 2122 1 2114 1 2106 1 2098 1 2090 1 2081 1 2064 1 2055 1 2046 1 2036 1 2214 1 2206 1 2199 1 2192 1 2184 1 2176 1 2168 1 2160 1 2152 1 2144 1 2135 1 2117 1 2108 1 2099
3. Section 2 Installation 2 2 1 UA 6 Detector and Optical Unit Preparation 2 2 2 Foxy R1 Fraction Collector 2 2 3 Retriever 500 Fraction Collector 2 2 4 Tube Piercer Preparation Optional Aperture Attaching the Flow Cell 2 2 The UA 6 detector and the Type 11 optical unit must be prepared as described in section 2 of the UA 6 manual 1 Install the pen and paper 2 The optical unit was shipped with a filter in place for UV detection at 254 nm If you desire a different wavelength refer to the section on Changing Wavelengths with the Type 11 Optical Unit The final preparation of the flow cell and optical unit is accom plished later in Section 2 2 4 of this manual The Foxy R1 fraction collector should be prepared according to section 2 of the Foxy R1 And R2 Fraction Collectors user manual 1 Perform the preliminary checkout procedure 2 Install the rack and tubes 3 Adjust the diverter valve height The Retriever 500 fraction collector should be prepared according to section 2 of the Retriever 500 Fraction Collector user manual 1 Perform the set up steps including automatic stop support rod installation and drop counter adjustment tubes should be during these steps 2 Perform the preliminary checkout procedure The tube piercer preparation requires a few steps Attach the flow cell to the top plate of the Tube Piercer Lastly attach the optical unit to the flow cell Typically the shipped flow
4. 1 2090 1 2269 1 2262 1 2254 1 2247 1 2239 1 2231 1 2223 1 2215 1 2206 1 2198 1 2189 1 2171 1 2162 1 2153 1 2144 1 2325 1 2318 1 2310 1 2302 1 2294 1 2286 1 2278 1 2270 1 2261 1 2253 1 2243 1 2225 1 2216 1 2207 1 2198 1 2381 1 2374 1 2365 1 2358 1 2349 1 2341 1 2333 1 2325 1 2316 1 2308 1 2298 1 2279 1 2270 1 2261 1 2252 a Calculated from equations developed by E J Barber in National Cancer Institute Monograph 21 June 1966 Density Gradient Fractionation System Appendix A Tables Table A 4 Centrifugal force chart for rotors commonly used in density gradient centrifugation Rotor Mfg Beckman Beckman IEC Beckman Beckman Beckman IEC Beckman Beckman Beckman IEC Beckman and SW25 1 SW25 2 SB 110 SW25 3 SW27 SW27 SB 206 SW 40 SW 41 SW 39L SB 405 SW 65L SB 283 SW 50L Tube Size diameter 5 8 5 8 1 14 5 9 16 9 16 1 2 10 9 length 4 4 3 1 2 96mm 3 3 4 3 1 2 2 54 7mm 1 069 1 098 1 131 1 160 1 125 1 051 1 111 1 062 685 704 4 276 4 394 4 525 4 640 4 500 4 203 4 444 4 249 2 739 2 817 9 622 9 886 10 181 10 440 10 125 9 457 9 999 9 559 6 163 6 339 17 105 17 575 18 100 18 560 18 000 16812 17 778 16 994 10 956 11 269 22 541 26 735 27 470 28 290 29 000 28 134 26 277 27 784 26 562 17 124 17 613 15 552 32 449 38 486 39 544 40 725 41 760 40 500 37 827 39 996 38 237 24 651 25 355 22 388 44 174 52 393 53 832 55 440 56 840 55 134 51 495 54 449 52 053 33 558 34 517 30 477 57 688 68 420 70 300 72 400 74
5. 1972 White clover mosaic 119 Berks R C M L A A B 41 June 1971 Wild cucumber mosaic 53 119 Fry et al Phytopathology 50 175 1960 Wound tumor 510 Black L M C M L A A B 34 Oct 1970 a Many viruses are multicomponent that is they have more than one virion like particles These particles may have different amounts of nucleic acid or no nucleic acid The particles are commonly referred to the sedimentation rates are usually given for the individual particles With rod shaped viruses multicomponent particles are referred to as short and long A 9 Density Gradient Fractionation System Appendix A Tables
6. 240 72 000 67 248 71 105 67 976 43 824 45 076 39 801 73 018 86 603 88 982 91 640 93 960 91 134 85 119 90 001 86 041 55 470 57 055 50 378 90 131 106 906 109 844 113 125 116 000 112 500 105 075 111 101 106 213 68 475 70 431 62 189 140 360 136 134 127 149 134 441 128 526 82 860 85 227 75 253 151 308 159 986 152 946 98 604 101 421 89 552 177 535 187 717 179 456 115 695 119 001 105 074 35 000 205 947 217 758 208 177 134 211 138 045 121 890 124 485 37 500 236 377 249 933 238 936 154 041 158 442 139 900 142 903 40 000 268 9929 284 419 271 904 175 396 180 304 159 203 162 592 42 500 197 893 203 518 179 725 183 551 45 000 221 867 228 197 201 492 205 781 47 500 247 195 254 229 224 506 229 280 50 000 273 910 281 725 248 755 52 500 310 574 274 257 55 000 340 887 300 994 57 500 372 553 328 536 60 000 405 684 258 207 62 500 388 685 65 000 420 396 Values given are for Maximum Radius of Rotor Maximum recommended speed for this rotor Maximum recommended speed for SB 283 and SW 39 rotors Maximum recommended speed for SW 50L rotor Maximum for A 841 rotor is 41 000 RPM Maximum recommended speed for SW 50L rotor Maximum for T 865 rotor ereaoop Density Gradient Fractionation System Appendix A Tables Table A 5 Sedimentation rates and ultraviolet absorbances of some representative viruses Virus w S20 Absorbance 1mg mL 1 cm at 260 nm Refer
7. 4 3 5 3 6 3 7 3 8 Density Gradient Fractionation System shown with Foxy R1 fraction collector 1 2 Attaching flow cell to tube piercer 2 3 System connections with a Foxy R1 fraction collector rotated view 2 5 System tubing connections with Foxy R1 fraction collector 2 6 System connections with a Retriever 500 fraction collector rotated view 2 9 System tubing connections with Retriever 500 fraction collector 2 10 Flow cell aperture installation 2 12 Collect fractions by volume counts 3 4 Cut peaks by peak widths and collect all fluids 3 5 Basic method settings 3 5 Piercing mechanism 3 7 Styrene butadiene rubber septum and collar set 3 7 Install the collar and ring 3 8 Inserting the centrifuge tube 3 10 Piercing the centrifuge tube 3 11 List of Tables 1 1 1 2 2 1 2 2 2 3 2 4 2 5 3 1 3 2 A 1 A 2 A 3 A 4 A 5 System Features oceng ea 2200 n ha ee durera ed eae be ben 1 3 System Specifications
8. Carnation latent Paul amp Welton Phytopath Z 49 401 1964 Carnation mottle Carnation ringspot Hollings amp Stone C M A A B 7 June 1970 Hollings amp Stone C M I A A B 21 Oct 1970 Carnation vein mottle Hollings amp Stone O W C M L A A B 78 Oct 1971 Cauliflower mosaic Shepherd R M C M A A B 24 Oct 1970 Cherry leaf roll Cropley amp Tomlinson C M A A B 80 Oct 1971 Chrysanthemum virus B Citrus leaf rugose virus 79 89 98 105 Hollings amp Stone C M I A A B 110 Oct 1972 Garnsey C M Gonsalves D C M A A B 164 Sept 1976 Citrus tristeza 140 10 Bar Joseph et al Phytopatholgy 60 75 1970 Clover yellow mosaic Cocksfoot mild mosaic 125 Bos L C M L A A B 111 July 1973 Huth amp Paul C M L A A B 107 Oct 1972 Cocksfoot mottle Catherall C M A A B 23 Oct 1970 Cowpea chlorotic A 6 Bancroft J B C M 1 A A B 49 June 1971 Density Gradient Fractionation System Appendix A Tables Table A 5 Sedimentation rates and ultraviolet absorbances of some representative viruses Continued w Virus S20 Absorbance 1mg mL 1 cm at 260 nm Reference Cowpea mosaic 58 95 115 6 2 10 0 8 1 Van Kammen C M I A A B 47 June 1971 Cucumber mosaic 98 5 0 Gibbs A J amp Harrison B D C M I A A B 1 June 1970 Cucumber necrosi
9. J E Hearst Progress in the Chemistry of Organic Natural Products XXI L Zechmeister ed Springer 1962 The density of Cs SO solutions may be calculated from the following formula Pos 1 0047 0 28369m 0 017428m 0 5 lt m lt 3 5 where mis the molality Ludlum and Warner J Biol Chem 240 2961 1965 c Specific gravity 20 4 C Density at 15 C Metrizamide is a trademark of Nyegaard amp Co Reg T M 3M Co A 3 Density Gradient Fractionation System Appendix A Tables Table A 3 Density of aqueous sucrose solutions g mL Temperature C Fraction Sucrose by Weight g mL Solution 0 0 20 4 0 6 0 8 0 10 0 12 0 14 0 16 0 18 0 1 0004 1 0004 1 0004 1 0004 1 0003 1 0002 1 0000 0 9998 0 9995 0 9992 0 9988 0 9980 0 9974 0 9969 0 9963 1 0043 1 0043 1 0043 1 0043 1 0041 1 0040 1 0038 1 0036 1 0033 1 0030 1 0026 1 0017 1 0012 1 0006 1 0000 1 0082 1 0082 1 0082 1 0081 1 0080 1 0079 1 0076 1 0074 1 0071 1 0068 1 0064 1 0055 1 0049 1 0044 1 0037 1 0122 1 0122 1 0122 1 0121 1 0119 1 0118 1 0115 1 0113 1 0109 1 0106 1 0102 1 0093 1 0087 1 0081 1 0075 1 0162 1 0162 1 0161 1 0160 1 0159 1 0157 1 0154 1 0152 1 0148 1 0145 1 0140 1 0131 1 0126 1 0120 1 0113 1 0203 1 0202 1 0202 1 0200 1 0199 1 0196 1 0194 1 0191 1 0187 1 0184 1 0179 1 0170 1 0164 1 0158 1 0152 1 0244 1 0243 1 0242 1 0241 1 0239 1 0236 1 0234 1 0231 1 0227 1 0223 1 0219 1 0209 1 0203 1 0197 1 0191 1 0285 1 0284 1 0283 1 0281 1 0277 1 0274 1 0271 1 0263 1 0258 1
10. M Note If the chart pen does not move to zero turn the BASELINE ADJUST control of the optical unit to MAXIMUM OPEN Move SENSITIVITY switch from SET LAMP amp OPTICS to desired absorbance range Align the RECORDER OFFSET control to its top center mark Push AUTO BASELINE The pen should deflect near mid scale M Note The baseline setting can remain near mid scale or moved to any arbitrary baseline by using the recorder offset control of the UA 6 detector Positioning the baseline will not alter the accuracy of the reading 10 Place the NOISE FILTER switch in the 1 5 position 11 Turn the PEAK SEPARATOR knob to 3 Refer to Section 3 of the UA 6 detector manual for detailed operating information At this time you should install a rack filled with tubes on the Foxy R1 fraction collector Refer to Section 2 of the Foxy R1 and Foxy R2 user manual After installing the rack adjust the height of the diverter valve if necessary The Foxy R1 fraction collector can now be configured and pro grammed for operation Refer to Configuration Settings in Section 3 of the fraction col lector user manual and configure the following settings 1 Touch the display to turn on the fraction collector 2 Ensure that the RACK 1 setting matches the installed rack and tubes Density Gradient Fractionation Systems Section 3 Operation 3 Set the TUBE ADVANCE SPEED to 2 4 Set the PUMP CONTROL to 0 This will briefly stop the
11. lists the tubing part number and the length you must cut from the bulk tubing supplied in the installation kit Connection types are identified by numbers in a triangle such as A Table 2 2 lists the hardware pieces from the installation kit and provides instruc tions to complete the connection Pieces of the connection hardware are identified by a lowercase letter such as a M Note Only finger tighten the plumbing fittings Never use a tool to tighten any system plumbing connection To user supplied waste container Figure 2 3 System tubing connections with Foxy R1 fraction collector 2 6 Density Gradient Fractionation Systems Section 2 Installation Table 2 1 Tubing and Lengths Tubing Fig 2 3 Cut Length Part Number Description As needed 023 0504 02 PTFE 0 062 ID 0 125 OD 8 200 mm 029 1304 02 Silicone 0 125 ID 0 25 OD for flow rates greater than 2 mL minute 029 1351 06 Silicone 0 065 ID 0 194 OD for flow rates less than 2 mL minute See Table 3 1 for recommended flow rate ranges 18 460 mm 023 0504 02 PTFE 0 062 ID 0 125 OD 4 100 mm 029 1304 02 Silicone 0 125 ID 0 25 OD 21 530 mm 023 0504 02 PTFE 0 062 ID 0 125 OD tubing volume app
12. panel connect the optical unit cable to the connector labeled OPTICAL UNIT Locate and connect the AC power adapter shipped with the Tris Pump a At the Tris pump front panel connect the 14 VAC con nector A CAUTION The AC power source must meet the requirements listed on the AC power adapter The factory ships Tris pump power adapters for North American or European power outlets For other locales it might be necessary to purchase a connector adapter from a local vendor b Connect the power adapter to an AC power source 8 The fraction collector and UA 6 detector were shipped with IEC power cords Use these to connect each instrument to an AC power source A CAUTION 2 8 Always refer to the instrument s serial number label for mains power requirements A CAUTION The instruments are shipped with either a North American 1EC320C13 to NEMA 5 15P power cord or a European 1EC320C13 to CEE7 VII power cord If the cord does not fit your AC mains power source purchase a connector adapter or 1EC320C13 power cord from a local vendor Density Gradient Fractionation Systems Section 2 Installation TOP Cable 60 1020 204 Figure 2 4 System connections with a Retriever 500 fraction collector rotated view 2 9 Density Gradient Fractionation Systems Section 2 Installation System plumbing 10 connections Figure 2 5 Move the Tris pump under the organizer shelf p
13. the col lar retaining nut 6 Loosely screw the collar retaining nut onto the flow cell to hold the collar and ring in place Fractionation is performed by pumping a chase liquid through the system The chase liquid gently pushes the contents of the centrifuge tube through the UV detector then into the fraction collection tubes Typically chase liquids are prepared just as the gradient solution except that the chase liquid must be a higher concen tration i e more dense See Appendix A for tables of chase liquids Adding dye to the chase liquid is a convenient method to ensure that the entire contents of the centrifuge tube have been delivered to the fraction collector The chase liquid must be more dense than the solution at the bottom of the gradient in the centrifuge tube Fluorinert FC 40 electronic liquid P N 68 0647 021 is a very satisfactory chase liquid for all common gradient materials Sucrose solutions are widely used as a chase liquid however sucrose solutions more concentrated than 1 8M 620 g L are too viscous to be forced through the small orifices of the system and should not be used Sucrose solutions may be used to chase sucrose glycerine Ficoll or dextran gradients but cannot be successfully used alone to chase dense solutions of salts such as NaBr or CsCl nor can the salts be used to chase sucrose Convection and disruption of the bottom of the gradient column occur if sugar solutions are chased with sa
14. 0254 1 0267 1 0248 1 0243 1 0237 1 0230 1 0326 1 0325 1 0324 1 0322 1 0320 1 0317 1 0314 1 0311 1 0307 1 0303 1 0298 1 0288 1 0282 1 0276 1 0270 1 0368 1 0367 1 0366 1 0361 1 0358 1 0355 1 0352 1 0348 1 0343 1 0339 1 0339 1 0328 1 0322 1 0316 1 0310 1 0411 1 0409 1 0407 1 0405 1 0403 1 0400 1 0396 1 0393 1 0389 1 0384 1 0380 1 0369 1 0363 1 0357 1 0350 1 0453 1 0452 1 0450 1 0447 1 0445 1 0441 1 0438 1 0434 1 0430 1 0426 1 0421 1 0410 1 0404 1 0397 1 0391 1 0496 1 0494 1 0492 1 0490 1 0487 1 0484 1 0480 1 0476 1 0472 1 0467 1 0462 1 0451 1 0445 1 0438 1 0432 1 0539 1 0537 1 0535 1 0532 1 0529 1 0526 1 0522 1 0518 1 0514 1 0509 1 0504 1 0492 1 0486 1 0480 1 0473 1 0583 1 0581 1 0578 1 0575 1 0572 1 0569 1 0565 1 0561 1 0556 1 0551 1 0546 1 0534 1 0528 1 0522 1 0515 1 0627 1 0625 1 0622 1 0619 1 0616 1 0612 1 0608 1 0603 1 0599 1 0594 1 0588 1 0577 1 0570 1 0564 1 0557 1 0671 1 0669 1 0666 1 0663 1 0659 1 0655 1 0651 1 0647 1 0642 1 0637 1 0631 1 0619 1 0613 1 0606 1 0599 1 0716 1 0713 1 0710 1 0707 1 0703 1 0699 1 0695 1 0690 1 0685 1 0680 1 0675 1 0662 1 0656 1 0649 1 0642 1 0761 1 0758 1 0755 1 0751 1 0747 1 0743 1 0739 1 0734 1 0729 1 0714 1 0718 1 0706 1 0699 1 0692 1 0685 1 0806 1 0803 1 0800 1 0796 1 0792 1 0788 1 0783 1 0778 1 0773 1 0768 1 0762 1 0749 1 0743 1 0736 1 0728 1 0852 1 0849 1 0845 1 0841 1 0837 1 0833 1 0828 1 0823 1 0818 1 0812 1 0806 1 0794 1 0787 1 0780 1 0772 1 0898 1 0894 1 0891 1 0887 1 0882 1 0878 1 0873 1 0868 1 0863 1 0857 1 0851
15. 1 0838 1 0831 1 0824 1 0816 1 0944 1 0940 1 0937 1 0933 1 0928 1 0923 1 0919 1 0913 1 0908 1 0902 1 0896 1 0883 1 0876 1 0869 1 0861 1 0991 1 0987 1 0983 1 0979 1 0974 1 0969 1 0964 1 0959 1 0953 1 0947 1 0941 1 0928 1 0921 1 0914 1 0906 1 1038 1 1034 1 1030 1 1025 1 1021 1 1016 1 1010 1 1005 1 0999 1 0993 1 0987 1 0973 1 0966 1 0959 1 0951 1 1085 1 1081 1 1077 1 1072 1 1067 1 1062 1 1057 1 1051 1 1045 1 1039 1 1033 1 1019 1 1012 1 1005 1 0997 1 1133 1 1129 1 1124 1 1119 1 1114 1 1109 1 1104 1 1098 1 1092 1 1086 1 1079 1 1065 1 1058 1 1051 1 1043 1 1181 1 1176 1 1172 1 1167 1 1162 1 1157 1 1151 1 1145 1 1139 1 1133 1 1126 1 1112 1 1105 1 1097 1 1089 1 1129 1 1225 1 1220 1 1215 1 1210 1 1204 1 1198 1 1192 1 1186 1 1180 1 1173 1 1159 1 1151 1 1144 1 1136 1 1278 1 1273 1 1268 1 1263 1 1258 1 1252 1 1246 1 1240 1 1234 1 1227 1 1221 1 1206 1 1199 1 1191 1 1183 1327 1376 1426 1476 1527 1578 1629 1680 1732 1784 1322 1 1317 1 1312 1371 1 1366 1 1361 1421 1 1416 1 1410 1471 1 1466 1 1460 1521 1 1516 1 1510 1572 1 1566 1 1560 1623 1 1617 1 1611 1674 1 1668 1 1662 1726 1 1720 1 1713 1778 1 1772 1 1765 1306 1355 1404 1454 1504 1554 1605 1656 1707 1758 1301 1 1295 1 1288 1 1282 1 1275 1 1268 1 1254 1 1246 1 1238 1 1230 1349 1 1343 1 1337 1 330 1 1323 1 1316 1 1302 1 1294 1 1286 1 1278 1398 1 1392 1 1386 1 1379 1 1372 1 1365 1 1350 1 1342 1 1334 1 1326 1448 1 1441 1 1435 1 1428 1 1421 1 1414 1
16. 1 2 x 2 1 2 Beckman Type 40 2 and 40 3 12 7 x 50 8 mm International 2865 12 7 x 98 4 mm International 0 375 to 0 750 9 16 x 3 1 2 Beckman SW 41 9 16 x 3 3 4 Beckman SW 40 14 5 x 96 mm International SB 283 and 206 14 5 x 102 mm 5 8 x 2 1 2 International Beckman 50 5 8 x 3 Beckman Type 40 TI 50 5 8 x 3 Beckman Quick Seal 342413 5 8 x 4 Beckman SW 25 3 16 1 x 76 2 mm International 495 23 x 70 mm MSE 23 ml 1x3 Beckman SW 25 1 1 50 to 3 0 1 x 3 1 2 Beckman SW 27 and Type 30 1 x 3 1 2 Beckman Quick Seal 342414 25 4 x 88 9 mm International SB 110 1 1 4 x 3 1 2 a If your centrifuge tube is not listed refer to a listed tube with the closest dimensions 3 2 Beckman SW 25 2 3 0 to 6 0 3 1 2 UA 6 Detector and Optical Unit 3 1 3 Foxy R1 Fraction Collector Foxy R1 Configuration Density Gradient Fractionation Systems Section 3 Operation Turn the PEAK SEPARATOR knob to the OFF position 2 Turn the CHART SPEED knob to OFF Place the STANDBY OPERATE switch in the OPERATE position Allow a minimum of 15 minutes warm up time to stabilize the lamp current 5 Set SENSITIVITY knob to SET LAMP amp OPTICS On the Optical Unit set the BASELINE ADJUST control to MAX OPEN Adjust the BASELINE ADJUST control of the optical unit until the chart pen moves to near zero
17. A geal ee ede ge II E 2 1 2 2 1 UA 6 Detector and Optical Unit Preparation 2 2 2 2 2 Foxy R1 Fraction Collector 2 2 2 2 8 Retriever 500 Fraction Collector 2 2 2 2 4 Tube Piercer Preparation 2 2 2 3 Installation of System with Foxy R1 Fraction Collector 2 4 2 4 Installation of System with Retriever 500 Fraction Collector 2 8 2 5 Changing the Flow Cell Aperture 2 12 Section 3 Operation 3 1 System Pr paration 2 ne n Gee se Nose caricia da Goda de oda idad 3 1 3 121 Tris Pumpin ci Aita 3 1 3 1 2 UA 6 Detector and Optical Unit 3 3 3 1 3 Foxy R1 Fraction Collector 3 3 3 1 4 Retriever 500 Fraction Collector 3 6 31 5 Tube Pierce a 3 6 3 1 6 Chase Liquid 2 2 062 050 cea de as See cee dira cea dune 3 9 3 2 Hractionations 4 402 25 a a A 3 10 3 3 Post Fractionation 3 13 3 4 Post Run Cleaning 0 A Late bones bleed ca Pe 3 13 Appendix A Tables A l Density Gradient Tables A 1 vii Density Gradient Fractionation Systems Table of Contents viii List of Figures 1 1 2 1 2 2 2 3 2 4 2 5 2 6 3 1 3 2 3 3 3
18. Density Gradient Fractionation Systems Installation and Operation Guide IAN TELEDYNE ISCO A Teledyne Technologies Company 69 3873 246 Copyright 2009 Teledyne Isco Inc Revision A February 18 2010 Foreword This instruction manual is designed to help you gain a thorough understanding of the operation of the equipment Teledyne Isco recommends that you read this manual completely before placing the equipment in service Although Teledyne Isco designs reliability into all equipment there is always the possi bility of a malfunction This manual may help in diagnosing and repairing the malfunc tion If the problem persists call or e mail the Teledyne Isco Technical Service Department for assistance Simple difficulties can often be diagnosed over the phone If it is necessary to return the equipment to the factory for service please follow the shipping instructions provided by the Customer Service Department including the use of the Return Authorization Number specified Be sure to include a note describing the malfunction This will aid in the prompt repair and return of the equipment Teledyne Isco welcomes suggestions that would improve the information presented in this manual or enhance the operation of the equipment itself Teledyne Isco is continually improving its products and reserves the right to change product specifications replacement parts schematics and instruc tions without notice Contact Informatio
19. I C M A A B 146 Oct 1975 Pepper veinal mottle Brunt amp Kenter C M 1 A A B 104 June 1972 Phalia cauliflower mosaic Brunt Virology 28 778 1966 Polio Schaffer amp Schwerdt Adv In Virus Res 6 159 1959 Popular mosaic Biddle C M L A A B 75 Oct 1971 Potato aucuba mosaic Kassanis B amp Govier D A C M I A A B 98 Oct 1972 Potato virus X Various Potato virus Y Delgado amp Grogan Phytopathology 56 1397 1966 A 7 Density Gradient Fractionation System Appendix A Tables Table A 5 Sedimentation rates and ultraviolet absorbances of some representative viruses Continued Virus Absorbance 1mg mL 1 cm at 260 nm Reference Potato Yellow Dwarf Brakke M K Virology 6 96 1958 Proteins Various Prunus necrotic ringspot 79 97 107 119 Fulton R W C M L A A B 121 July 1973 Radish mosaic T 57 M 97 B 116 Campbell R N C M A A B 121 July 1973 Red clover mottle 60 101 127 Gibbs et al Ann Appl Biol 61 99 1968 Red clover vein mosaic 160 Varma Anupam C M A A B 22 Oct 1970 Ribonucleic acid Various Rice dwarf Rice tungro Rice yellow mottle Lider et al C M I A A B 102 Oct 1972 Galvez G E C M I A A B 67 Oct 1971 Bakker U C M A A B 149 Oct 1975 Rod shaped viruses Various Saguaro cactus viru
20. Systems Section 1 Introduction Table 1 1 System Features Name and Description The Tris Peristaltic Pump pumps the chase liquid 60 3877 060 Tube Piercer Stand accommodates most common centrifuge tubes The stand allows you to upwardly displace the gradient and material This is done by directing a chase solution through a hole pierced in the tube bottom 60 0084 054 A Density Gradient Flow Cell is mounted at the top of the tube piercer The system ships with a 5 mm flow cell A 2mm flow cell option is available 68 1140 006 The Optical Unit is also mounted above the tube piercer The optical unit can be configured for either 254 or 280 nm wavelengths using the supplied filters 68 3870 011 or 68 3880 001 The Fraction Collector Foxy R1 shown provides hands off fraction collection Peaks are cut as new fractions to isolate UV absorbing material The Foxy R1 fraction collector includes a 1 5 mL microcentrifuge tube collec tion rack The Retriever 500 fraction collector includes tube racks for 10 to 16 mm tubes 68 0940 016 UA 6 Detector to detect UV absorbing material passing through the flow cell The UA 6 includes a chart recorder 7 69 2183 001 The Organizer Shelf conserves bench space Items Not shown Interface Cables 69 2134 172 or 60 1020 204 69 2134 173 or 60 1020 217 Foxy R1 to Tris pump Retriever 500 to Tris pump Foxy R1 to UA 6 detector Retriever 500 to UA 6 detec
21. Tris pump during tube changes 5 Verify that the ANALOG PEAK setting is at 1000 mV Foxy R1 Programming The fraction collector uses a programmed method to control the fractionation and peak cutting The method also delays the arm movement so that tube advances occur when material observed by the detector is present at the drop former Refer to Method Settings in Section 3 of the fraction collector user manual and program the following nominal method which assumes e 1 mL fractions will be collected in 1 5 mL microcen trifuge tubes e a21linch length of PTFE 0 062 inch ID tubing installed between the optical unit and diverter valve tubing E e a Y16 inch ID tube is in the Tris Pump To program the fraction collector 1 Program a method to collect fractions by volume counts from the Tris pump a From the main display touch the FOLDER icon and then the VOLUME icon to fractionate by fixed volumes Figure 3 1 ep Figure 3 1 Collect fractions by volume counts b Ensure that the TUBE icon is active highlighted by a surrounding box If not touch the icon to toggle the selection c Enter the volume count as determined by the ID of tub ing in the Tris pump With Y8 inch ID tubing the pump sends about 22 counts for each milliliter with 1 inch tubing the pumps sends about 78 counts for each milliliter For this example enter 78 d Touch the DELAY hourglass icon and enter a delay volume of 78 counts T
22. Un thread the headless nut and verify that 1 the yellow ferrule is flush and perpendicular with the end of the tubing 2 the metal lock ring is compressed over the ferrule without any gaps and 3 all pieces are correctly aligned and free from any deformation If not fully swaged reinsert the headless nut into the port and tighten further Un thread the headless nut and inspect again using this criteria 2 7 Density Gradient Fractionation Systems Section 2 Installation 2 4 Installation of System 1 with Retriever 500 Fraction Collector 2 3 Interface cable connections 4 Figure 2 4 Power source connections 7 Figure 2 4 Position system components on the organizer shelf as shown in Figure 2 4 Ensure that the UA 6 detector power switch is in the OFF position Set the Tris pump CCW STOP CW switch to the STOP position Locate cable 60 1020 217 that was shipped with the sys tem components a At the back panel of the fraction collector connect the 8 pin DIN connector to the connector labeled DETEC TOR b At the back panel of the UA 6 detector connect the 9 pin connector labeled Fraction Collector Locate cable 60 1020 204 that was shipped with the sys tem components a At the back panel of the fraction collector connect the 6 pin DIN connector to the connector labeled PUMP b At the front panel of the Tris pump connect the cable to the connector labeled SIGNAL At the UA 6 detector back
23. adient Fractionation Systems Section 2 Installation 2 3 Installation of System 1 with Foxy R1 Fraction Collector 2 3 Interface cable connections 4 Figure 2 2 Power source connections 7 Figure 2 2 Position system components on the organizer shelf as shown in Figure 2 2 Ensure that the UA 6 detector power switch is in the OFF position Set the Tris pump CCW STOP CW switch to the STOP position Locate cable 69 2134 173 that was shipped with the sys tem components a At the back panel of the fraction collector connect the 8 pin mini DIN connector to the connector labeled DETECTOR b At the back panel of the UA 6 detector connect the 9 pin connector labeled FRACTION COLLECTOR Con nect the banana plug connectors between the GROUND center and the 1V Recorder connections Locate cable 69 2134 172 that was shipped with the sys tem components a At the back panel of the fraction collector connect the 6 pin mini DIN connector to the connector labeled PUMP b At the front panel of the Tris pump connect the cable to the connector labeled SIGNAL At the UA 6 detector back panel connect the optical unit cable to the connector labeled OPTICAL UNIT Locate and connect the AC power adapter shipped with the Tris Pump a At the Tris pump front panel connect the 14 VAC con nector A CAUTION 2 4 The AC power source must meet the requirements listed on the AC power adapter The fact
24. ceed the chart scale adjust the sensitivity as needed An LED indicator on the front panel also indicates the current peak state e The LED is off as the chart continues along the baseline zero slope e During a positive slope the LED is green e At the maximum of the peak zero slope the LED is off e As the slope returns toward the baseline with downward slope the LED is red e The LED turns off when the slope returns to zero This might not be at the original baseline Peak cutting The fraction collector will perform a tube advance at the beginning and end of a peak to isolate peak mate rials from non peak The Retriever 500 will perform these tube advances when the LED on the UA 6 changes from off to green and when it changes from red to off The Foxy R1 was pro grammed for internal peak detection with a delay volume Because the peaks are being cut much closer to when the material is present at the drop former these tube advances will not coincide with the LED on the UA 6 detector Stop the run when the all of the contents of the density gradient tube have been pushed to the fraction collector 1 At the Tris pump place the CCW OFF CW switch in the OFF position 2 Stop the fraction collector Foxy R1 touch the STOP icon Retriever 500 push the RUN STOP button 3 At the UA 6 detector set the CHART SPEED to OFF 3 3 Post Fractionation 3 4 Post Run Cleaning Density Gradient Fractionation Systems Sectio
25. cell has a 2 or 5 mm optical path length and a 1 mm height aperture This combination is suitable for many density gradient applications and no changes should be necessary The flow cell s pathlength was selected when the system was ordered If during your initial runs you find that the materials of interest absorb very little UV light you can choose to illuminate a larger volume refer to Section 2 5 to install a 2 8 mm aperture 1 Attach the flow cell to the tube piercer Figure 2 1 a Remove the collar retaining nut from the flow cell b Remove the locking nut from the flow cell c Insert the flow cell into the top plate of the tube piercer d Reinstall the locking nut and hand tighten e Reinstall the collar retaining nut and hand tighten Density Gradient Fractionation Systems Section 2 Installation Flow Cell C Optical Unit Notch Top Plate of Tube Piercer Flow Cell Locking Nut Collar Retaining Nut Figure 2 1 Attaching flow cell to tube piercer 2 Attach the optical unit around the flow cell a The front face of the optical unit has two white catches Release the catch on the left side measuring cell side This will allow the optical unit to swing open b Note the notch near the base of the flow cell Hold the optical unit so that the case bottom will fit in this notch and then close the optical unit c Secure the optical unit with the catch 2 3 Density Gr
26. cer a Loosen the rear thumbscrew and lower the tube pierc ing mechanism b Fully insert the tube into the top assembly through the collar retaining nut Figure 3 7 c Hand tighten the collar retaining nut to seal the tube A CAUTION Do not over tighten the nut Over tightening the nut may deform the tube wall or tear the collar resulting in leaks The nut should be only snug d Ensure that the tube is vertically aligned If not push the tube to align it e Raise the bottom tube piercing mechanism until the septum is depressed by about 5 mm 3 16 inch against the spring f Tighten the thumbscrew to secure the tube 1 Insert tube and tighten 2 Loosen rear thumbscrew 3 Raise the tube piercing collar retaining nut mechanism and tighten thumbscrew Figure 3 7 Inserting the centrifuge tube 3 10 Density Gradient Fractionation Systems Section 3 Operation M Note If you are fractionating a 1 4 inch diameter tube place the O ring seal around the top edge of the tube Next remove the collar retaining nut and insert the tube so it is centered in the assembly Replace the collar retaining nut and hand tighten to seal the tube Then raise the tube piercing mechanism and tighten the thumbscrew 2 Pierce the tube A CAUTION Tubes made of materials such as polycarbonate and glass cannot be pierced a Turn the bottom cap to raise the needle Figure 3 8 b Continue to turn the bottom cap whil
27. e insert heights and path lengths Table 2 5 Flow Cell Illuminated Volumes Light Aperture Path Length Illuminated Volume Height mm mm pl 2 12 Density Gradient Fractionation Systems Section 3 Operation 3 1 System Preparation 3 1 1 Tris Pump 0 375 mL min 1 50 mL min This section describes the general operation of the Density Gra dient Fractionation system The steps and system settings are intended to serve as the basis for a fractionation method which should be empirically modified for your application Please refer to the individual component manuals for additional operating instructions Both models of fraction collectors are discussed in this section When following instructions relate to fraction collectors perform only those instructions for the installed model This section covers the preliminary system settings for basic fractionation operation and priming the inlet tubing with a chase liquid The maximum flow rate of the chase liquid is limited by a number of factors e If a constant speed recorder is used it may give a compressed scanning curve for high flow rates resulting in poorer apparent resolution of peaks e Ifa fast flow rate is used with very viscous solutions pressure may build up in the system and leaks may occur e Turbulence or laminar flow may occur at higher flow rates causing a decrease in resolution observed at the absorbance monitor e The recorder pen move
28. e watching the bottom of the tube The tip of the needle will pierce the tube c Stop turning the bottom cap when the holes in the nee dle tip are just inside the tube Figure 3 8 Piercing the centrifuge tube 3 Start the UA 6 detector a Uncap the pen b Select the SENSITIVITY This setting should be the expected absorbance If unknown use 0 2 as an initial value c Adjust the RECORDER OFFSET for the desired pen position d Select the desired CHART SPEED A setting of 15 cm hr is a suggested nominal value Refer to the UA 6 and Tris pump manuals for more information about chart speed 4 At the Tris pump place the CCW OFF CW switch in the CW position The chase liquid will begin filling the centri fuge tube Density Gradient Fractionation Systems Section 3 Operation 3 12 Stopping the Fractionation Run 5 Start the fraction collector Foxy R1 touch the PLAY icon Retriever 500 push the RUN STOP button The system will begin to fractionate the centrifuge tube contents During operation you can observe the following Chase liquid If a dye was added to the liquid you can observe the dense chase liquid displacing the gradient Fractionation The fraction collector will perform a tube advance at the set fractionation interval Using these nominal program settings the fraction collector will change tubes every 1 mL approximate Peak detection The UA 6 detector charts the absorbance If peaks ex
29. ence Alfalfa mosaic Tz 53 To 60 Ta 68 Tb 76 M 89 B 99 5 2 Bos L amp Jaspers E M J C M A A B 46 June 1971 Apple chlorotic leaf spot 96 Lister R M C M L A A B 30 Oct 1970 Apple mosaic 88 117 Fulton R W C M L A A B 83 June 1972 Arabis Mosaic 129 Harrison amp Nixon Virology 12 104 1960 Barley stripe mosaic 185 Atebekov J G amp Novikov V K C M I A A B 68 Oct 1971 Barley yellow dwarf 117 Rochow amp Brakke Virology 24 310 1964 Bean pod mottle 54 91 112 Semancek J S C M L A A B 108 Oct 1972 Belladonna Black raspberry latent Broad bean mottle 53 113 81 89 98 84 8 Paul H L C M A A B 52 June 1971 Lister amp Converse C M I A A B 106 Oct 1972 Gibbs A J C M I A A B 101 Oct 1972 Broad bean stain 60 100 127 Gibbs amp Smith C M A A B 29 Oct 1970 Broad bean true mosaic 98 119 Paul H L C M A A B 20 June 1970 Broad bean wilt 63 100 126 Taylor amp Stubbs C M A A B 81 June 1972 Broccoli necrotic yellows Brome mosaic 874 41 Campbell amp Lin C M A A B 85 June 1972 Bockstahler amp Kaesburg J Biophys 2 1 1962 Brakke M K unpublished data Cacao swollen shoot Brunt A A C M A A B 10 June 1970 Cacao yellow mosaic Brunt A A C M A A B 11 June 1970
30. he approximate volume of the tubing from the optical unit to the diverter valve is 1 mL Table 2 1 3 4 Density Gradient Fractionation Systems Section 3 Operation e Touch the ENTER icon to save the settings and return to the method settings display 2 Program the method to cut peaks based on the peak width of the analog signal of the UA 6 detector a Touch the FOLDER icon and then the PEAK WIDTH icon wW Figure 3 2 Cut peaks by peak widths and collect all fluids b Enter a peak width of 2 minutes c The peak width icon should be shaded below the peak This indicates that the fraction collector will collect all fluids instead of diverting non peak fluids to waste Touch the icon to toggle the state until the icon is shaded d Touch the ENTER icon to save the settings and return to the method settings display 3 From the method settings display verify that the only the VOLUME and PEAK WIDTH options are active as shown in Figure 3 3 If not disable the other active features Figure 3 3 Basic method settings 4 Touch the ENTER icon to return to the main display 3 5 Density Gradient Fractionation Systems Section 3 Operation 3 6 3 1 4 Retriever 500 Fraction Collector 3 1 5 Tube Piercer Septum Installation The fraction collector is now programmed with a basic collection method The Foxy R1 fraction collector has many advanced fea tures that may be included in the method
31. hen you are finished with all fractionation runs for the day This will prevent material from collecting or crystallizing in the liquid path 1 Remove the chase liquid container Cap and store if desired 2 Remove all tubing 3 Remove the optical unit from the top of the tube piercer A CAUTION Do not immerse any electronic instrument component Density Gradient Fractionation Systems Section 3 Operation 4 Remove the flow cell and upper assembly from the tube piercer stand Separate the flow cell from the upper assem bly 5 Remove the bottom tube piercing mechanism from the tube piercer stand This is held in place by the front thumb screw 6 Wash the tubing tubing connectors flow cell upper assem bly tube piercing mechanism with needle Generally hot water and a mild detergent is sufficient M Note The flow cell windows do not normally need to be removed for cleaning M Note If the piercing needle becomes clogged it may be cleaned with the piece of 22 gauge 0 029 inch diameter wire supplied with the tube piercer 7 Clean any spillage of solution on the instruments and shelf 8 Reassemble the system when the parts are clean and dry 3 14 Density Gradient Fractionation System Appendix A Tables A 1 Density Gradient The following tables are applicable to typical Density Gradient Tables Fractionation applications Table A 1 Viscosity of various salt solutions used for density gradien
32. into the bottom of the centrifuged tube raising the gra dient intact by bulk flow Chase solution is injected by piercing the bottom of the tube A CAUTION Tubes made of materials such as polycarbonate and glass cannot be pierced AN WARNING Avoid hazardous practices If you use this instrument in any way not specified in this manual the protection provided by the instrument may be impaired this will increase your risk of injury Density Gradient Fractionation systems are available with Teledyne Isco s Foxy R1 or Retriever 500 fraction collectors Refer to Figure 1 1 and Table 1 1 for an overview of the features of both systems The system with the Retriever 500 fraction collector collects all fluids from the fractionation run and can cut peaks using the slope based peak separation feature of the UA 6 detector The system with the Foxy R1 fraction collector provides advanced peak detection and collection options The fraction col lector can analyze the analog peak signal from the UA 6 detector and cut fractions using threshold slope time windows or a com bination of these options A programmable diverter valve can collect all fluids or divert non peak fluids to a waste container Density Gradient Fractionation Systems Section 1 Introduction Figure 1 1 Density Gradient Fractionation System shown with Foxy R1 fraction collector Item No Fig 1 1 Catalog Number 68 1610 010 Density Gradient Fractionation
33. l SB 405 1 2x2 Beckman SW 39 50 65 50 1 1 2 x 2 1 2 Beckman Type 40 2 and 40 3 12 7 x 50 8 mm International 2865 12 7 x 98 4 mm International 9 16 x 3 1 2 Beckman SW 41 9 16 x 3 3 4 14 5 x 96 mm Beckman SW 40 International SB 283 and 206 14 5 x 102 mm International 5 8 x 2 1 2 Beckman 50 5 8 x 3 Beckman Type 40 TI 50 5 8 x 4 16 1 x 76 2 mm 23x 70 mm Beckman SW 25 3 International 495 MSE 23 ml U JO JUO OOO O U OU U O0 gt gt F Collar E Ring 1x3 Beckman SW 25 1 E 1 x 3 1 2 Beckman SW 27 and Type 30 25 4 x 88 9 mm International SB 110 1 1 4 x 3 1 2 1 2x 2 5 8 x 3 Beckman SW 25 2 Beckman Quick Seal 342412 Beckman Quick Seal 342413 G Collar F Ring G 1x 3 1 2 Beckman Quick Seal 342414 a If your centrifuge tube is not listed simply select the best fitting collar Flow Cell Collar Ring Collar Retaining Nut Figure 3 6 Install the collar and ring 3 8 H 3 1 6 Chase Liquid Density Gradient Fractionation Systems Section 3 Operation 2 Cut the collar from the septum and collar set Figure 3 5 Use a sharp knife and trim excess material from the cut edge 3 Remove the collar retaining nut from the piercing mecha nism Figure 3 6 4 Insert the collar into the flow cell 5 Insert the stainless steel ring into the groove inside
34. le standards and test results The maximum altitude rating is per European Norm 61010 1 which establishes safety requirements for elec trical equipment The rating pertains to electrical creepage and clearances The altitude rating is not applicable to system performance 1 3 About this Manual 1 4 For Additional Information 1 4 This manual covers the installation and operation of Density Gradient Fractionation systems with both available fraction col lectors While completing the installation and operation steps that relate to a fraction collector complete only the sections for the fraction collector you received with your system For example if you received the Foxy R1 fraction collector with your system skip all instructions that relate to the Retriever 500 fraction collector Complete descriptions and operating instructions are beyond the scope of this installation guide Refer to the instruction manuals supplied in the system manual for the individual components Contact your local sales representative for further assistance with the Density Gradient Fractionation system Density Gradient Fractionation Systems Section 2 Installation 2 1 Unpacking 2 2 System Assembly This section will cover setup of Isco Density Gradient Fraction ation systems with the Foxy R1 and Retriever 500 fraction col lectors including the connection of electrical and interface cables and system plumbing The system is shipped in mul
35. lt solutions or vice versa This convection is apparently a result of the widely different diffusion rates of salt and sucrose and the resulting loss of salt from the salt solution next to the sucrose solution The use of Fluorinert FC 40 is highly recom mended in all cases After selecting a chase liquid 1 Prepare more than enough chase liquid to fill the centri fuge tube volume as well as the volume of system tubing This will ensure that the pump will deliver the full gradi ent to the fraction collector and leave the system primed for the next run 2 Place the Tris pump s inlet tubing into the chase liquid container 3 Place an absorbent wipe over the Tube Piercer needle opening 3 9 Density Gradient Fractionation Systems Section 3 Operation 4 Pump chase liquid until the liquid just reaches the Tube Piercer needle opening To do so a Place the CCW OFF CW switch in the CW position The pump begins to operate Observe the flow of chase liq uid through the pump and up to the Tube Piercer b When the chase liquid reaches the needle opening place the CCW OFF CW switch in the OFF position M Note If there is air in the tubing allow the pump to continue to run to clear the air bubbles Air bubbles may disturb the material sus pended in the gradient and contribute noise to peak detection 3 2 Fractionation To fractionate a density gradient centrifuge tube 1 Insert a tube lt 1 inch diameter into the Tube Pier
36. n Customer Service Phone 800 228 4373 USA Canada Mexico 402 464 0231 Outside North America Fax 402 465 3022 Email IscoCSR teledyne com Technical Support Phone 800 775 2965 Analytical 866 298 6174 Samplers and Flow Meters Email IscoService teledyne com Return equipment to 4700 Superior Street Lincoln NE 68504 1398 Other Correspondence Mail to P O Box 82531 Lincoln NE 68501 2531 Email IscoInfo teledyne com Web site www isco com Revised March 17 2009 General Warnings Density Gradient Fractionation Systems Safety Before installing operating or maintaining this equipment it is imperative that all hazards and preventive measures are fully understood While specific hazards may vary according to location and application take heed of the following general z je iat D m 5 Ue n A WARNING Liquids associated with this instrument may be classified as carcinogenic biohazard flammable or radioactive Should these liquids be used it is highly recommended that this application be accomplished in an isolated environment designed for these types of materials in accordance with federal state and local regulatory laws and in compliance with your company s chemical hygiene plan in the event of a spill AVERTISSEMENT Eviter de r pandre des liquides dangereux Les liquides qui sont analys s dans cet instrument peuvent tre canc rig nes hasards biologiques inflammables ou radi
37. n 3 Operation After stopping the fractionation run the fractions may be removed from the fraction collector The following steps should be performed to remove the centrifuge tube and prepare the system for the next fractionation run 1 At the Tris pump place the CCW OFF CW switch in the CCW position This removes the chase liquid from the tub ing M Note The Tris pump CONTROL knob may be turned to 100 to expedite this step 2 When the chase liquid has withdrawn to a point just above the piercing needle place the CCW OFF CW switch in the OFF position This small volume above the needle ensures that air will not be in the needle for the next run M Note If the Tris pump CONTROL knob was turned to 100 return it to the original setting now 3 Loosen the rear thumbscrew and lower the tube piercing mechanism 4 Loosen the collar retaining nut 5 Remove the centrifuge tube from the tube piercer Clean up excess chase liquid 6 Load the fraction collector with empty tubes 7 Very small amounts of chase liquid will be lost at the end of the run and when the centrifuge tube is removed Replen ish the chase liquid container if necessary Section 3 1 6 8 Review the operation settings of the pump detector and fraction collector Revise the settings as necessary to opti mize the system for your application The system is ready for the next fractionation run Refer to Section 3 2 The system should be cleaned w
38. n Systems Section 2 Installation Table 2 3 Tubing and Lengths Tubing Fig 2 3 Cut Length Part Number Description As needed 029 1304 02 Silicone 0 125 ID 0 25 OD 8 200 mm 029 1304 02 Silicone 0 125 ID 0 25 OD for flow rates greater than 2 mL minute 029 1351 06 Silicone 0 065 ID 0 194 OD for flow rates less than 2 mL minute See Table 3 1 for recommended flow rate ranges 32 813 mm 023 0502 04 FEP 0 030 ID 0 0625 OD 3 76 mm 029 1304 02 Silicone 0 125 ID 0 25 OD 16 406 mm 023 0502 04 FEP 0 030 ID 0 0625 OD tubing volume approximately 0 8 mL Table 2 4 Connection Hardware E Connection Diagram Item Descriptions and Instructions a Connector Nut 60 0923 015 b 1 8 Ferrule 60 0923 017 c Barbed Connector 60 1613 112 Instructions Place Ferrule narrow end first in Barbed Connector Loosely thread Connector Nut onto Barbed Connector Insert PTFE tubing into Connector Nut and finger tighten Push silicone tubing over barbed end Connector Nut 60 0923 015 1 8 Ferrule 60 0923 017 Black Lead Connector 60 0923 013 Flow cell on Brandel tube piercer Instructions 1 Thread Lead Connector into Flow Cell 2 Place Ferrule narrow end first in Lead Connector 3 Loosely thread Connector Nut onto Lead Connector 4 Insert PTFE tubing into Connector Nut and finger tighten a Connecto
39. oactifs Si vous devez utiliser tels liquides il est tres recommand que vous le faites a l int rieur d un environnement isol concu pour tels liquides Cet environnement isol devrait tre construit selon les reglements f d raux provinciaux et locaux aussi que le plan de votre compagnie qui concerne l v nement d un accident avec les mati res hasardeuses AN WARNING Avoid hazardous practices If you use this instrument in any way not specified in this manual the protection provided by the instrument may be impaired A AVERTISSEMENT Eviter les usages p rilleux Si vous utilisez cet instrument d une mani re autre que celles qui sont specifi es dans ce manuel la protection fournie de l instrument peut tre affaiblie cela augmentera votre risque de blessure Density Gradient Fractionation Systems Safety Hazard Severity Levels Ay WARNING If this system uses flammable organic solvents Teledyne Isco recommends that you place this system in a well ventilated environment designed for these types of materials This environment should be constructed in accordance with federal state and local regulations It should also comply with your organization s plan concerning chemical and hygiene mishaps In all cases use good laboratory practices and standard safety procedures A AVERTISSEMENT Ce syst me peut utiliser des dissolvants organiques inflammables Pour r duire le p ril qui peut tre caus par l acc
40. ory ships Tris pump power adapters for North American or European power outlets For other locales it might be necessary to purchase a connector adapter from a local vendor b Connect the power adapter to an AC power source 8 The fraction collector and UA 6 detector were shipped with IEC power cords Use these to connect each instrument to an AC power source AN CAUTION Always refer to the instrument s serial number label for mains power requirements Density Gradient Fractionation Systems Section 2 Installation TOP Figure 2 2 System connections with a Foxy R1 fraction collector rotated view 2 5 Density Gradient Fractionation Systems Section 2 Installation A CAUTION The instruments are shipped with either a North American 1EC320C13 to NEMA 5 15P power cord or a European 1EC320C13 to CEE7 VII power cord If the cord does not fit your AC mains power source purchase a connector adapter or 1EC320C13 power cord from a local vendor 9 Move the Tris pump under the organizer shelf positioning it towards the front as shown in Figure 1 1 on page 1 2 This will allow you to plumb the system from the front System plumbing 10 Refer to Figure 2 3 Table 2 1 and Table 2 2 and complete connections the plumbing connections Figure 2 3 When using the figure and tables note the following Each piece of tubing is identified by an uppercase letter in a circle such as A Table 2 1
41. ositioning it towards the front as shown in Figure 1 1 on page 1 2 This will allow you to plumb the system from the front Refer to Figure 2 5 Table 2 3 and Table 2 4 and complete the plumbing connections When using the figure and tables note the following Each piece of tubing is identified by an uppercase letter in a circle such as A Table 2 1 lists the tubing part number and the length you must cut from the bulk tubing supplied in the installation kit Connection types are identified by numbers in a triangle such as A Table 2 4 lists the hardware pieces from the installation kits and provides instruc tions to complete the connection Pieces of the connection hardware are identified by a lowercase letter such as a Only finger tighten the plumbing fittings Never use a tool to tighten any system plumbing connection l RETRIEVER 500 DISPLAY MODE ours n use omea onn vo o e ost ne orme 1 un o D Y g 03 Figure 2 5 System tubing connections with Retriever 500 fraction collector 2 10 Density Gradient Fractionatio
42. r Nut 60 0923 015 b 1 8 Ferrule 60 0923 017 c Red Lead Connector 60 0643 254 d Drop former on Retriever 500 Instructions 1 Thread Lead Connector into Drop Former 2 Place Ferrule narrow end first in Lead Connector 3 Loosely thread Connector Nut onto Lead Connector 4 Insert PTFE tubing into Connector Nut and finger tighten Density Gradient Fractionation Systems Section 2 Installation 2 5 Changing the Flow The flow cell was shipped from the factory with 1 0 mm aper Cell Aperture tures installed A larger 2 8 mm aperture set is in the accessory kit should you need to illuminate a larger volume To change the flow cell apertures 1 On one side of the flow cell remove the window nut using the wrench included with the flow cell Figure 2 6 2 Remove the 1 0 mm aperture 3 Verify that the O ring is in place 4 Insert the 2 8 mm aperture Position the aperture so that the opening is horizontal 5 Install the window nut and tighten 6 Repeat steps 1 through 5 for the other side 7 Verify that both light aperture slits are in the horizontal plane as shown in Figure 2 6 If not loosen the window nuts align the openings and tighten the window nuts Main Bod Light Apertur S Window Nut DN Wrench on Figure 2 6 Flow cell aperture installation Slit in horizontal plane M Note The illuminated volumes are specified in Table 2 5 as well as the light apertur
43. ring and collar to secure the top of the tube To install a septum 1 Turn the bottom cap of the piercing mechanism Figure 3 4 to lower the piercing needle 2 Cut a septum from the septum and collar set Figure 3 5 Use a sharp knife and cleanly trim any excess material from the cut edge 3 Insert the septum over the spring in the piercing mecha nism Density Gradient Fractionation Systems Section 3 Operation Septum Bottom Holder Center y Column s amp Rear Thumbscrew Bottom Cap Front Inlet Thumbscrew Figure 3 4 Piercing mechanism Figure 3 5 Styrene butadiene rubber septum and collar set Ring and Collar A ring and collar secures and seals tubes lt 1 inch diameter in Installation the top assembly To install a ring and collar 1 Determine the size of centrifuge tube and select the collar and ring according to Table 3 2 M Note If you have selected a 1 1 4 inch diameter tube skip the ring and collar installation steps These larger diameter tubes do not use a ring and have an O ring type collar which is installed at the time the tube is loaded step 1 note section 3 2 3 7 Density Gradient Fractionation Systems Section 3 Operation Centrifuge Tube Size Table 3 2 Collar and Ring Selection Mfg Rotor Designation Collar amp Ring Designation 7 16 x 1 15 16 Beckman A 7 16 x 2 3 8 Beckman SW 56 10 9 x 54 7 mm Internationa
44. roximately 1 mL As needed 023 0504 02 PTFE 0 062 ID 0 125 OD Table 2 2 Connection Hardware Union Fig 2 3 Connection Diagram Item Descriptions and Instructions a Connector Nut 60 0923 015 b 1 8 Ferrule 60 0923 017 c Barbed Connector 60 1613 112 Instructions Place Ferrule narrow end first in Barbed Connector Loosely thread Connector Nut onto Barbed Connector Insert PTFE tubing into Connector Nut and finger tighten Push silicone tubing over barbed end Connector Nut 60 0923 015 1 8 Ferrule 60 0923 017 Black Lead Connector 60 0923 013 Flow cell on Brandel tube piercer Instructions 1 Place Ferrule narrow end first in Lead Connector 2 Loosely thread Connector Nut onto Lead Connector 3 Insert PTFE tubing into Connector Nut and finger tighten a Ferrule and Locking Ring 209 0163 21 Note Tapered edge of locking ring must face the ferrule b Headless Nut 209 0163 24 Instructions 1 Slide Nut onto PTFE tubing 2 Slide Locking Ring and Ferrule onto PTFE tubing Note Tapered edge of locking ring must face the ferrule 3 Push PTFE tubing fully into port on back of diverter valve 4 Finger tighten Nut to swage Locking Ring and Ferrule Refer to the Foxy R1 instruction manual for correct routing and identification of inlet and waste ports b An inspection of the swaged fitting is recommended
45. s Satellite virus Scrophularie 50 169 231 332 T54 B 116 T amp B 8 Nelson M R amp Tremaine J R C M L A A B 148 Oct 1975 Kassanis C M 1 A A B 15 June 1970 Bercks R C M L A A B 113 July 1973 Shope papilloma 280 Schachmann H K J Am Chem Soc 73 4453 Simian virus 40 240 Black et al Virology 24 381 1964 Soil borne wheat mosaic 172 211 Brakke M C M A A B 77 Oct 1971 Southern bean mosaic Sowbane mosaic Spherical virus 115 Various Kodo C I C M I A A B 64 Oct 1971 Various Squash mosaic T57 M95 B 118 Campbell R N C M A A B 43 June 1971 Sugarcane mosaic 175 55 168 65 155 3 148 25 Various Sunn hemp mosaic 20 50 70 80 187 Kassanis B 8 Varma A C M L A A B 153 Oct 1975 To T4 Tg Phages 700 900 1000 Cummings D J Virology 23 408 1964 Hook A I et al J Biol Chem 165 241 1966 T3 B Phage 470 Cummings D J Virology 23 408 1964 T3 C Phage 366 Cummings D J Virology 23 408 1964 T 5 Phage T 7 Phage 469 606 Cummings D J Virology 23 408 1964 Cummings D J Virology 23 408 1964 Tobacco etch A 8 Purcifull Virology 29 8 1964 Density Gradient Fractionation System Appendix A Tables Table A 5 Sedimentation rates and ultraviolet absorbances of some representative viruses Con
46. s 113 Dias amp Doanne Can J Bot 46 47 1968 Dahlia mosaic 254 Brunt A A C M A A B 51 June 1971 Echtes Ackerbohnenmosaik 98 119 Gibbs A J amp Paul H L C M L A A B 20 June 1970 Foot and mouth disease 140 Bachrach H L Virology 25 532 1965 Grapevine chrome mosaic 92 117 Marletti C P C M L A A B 103 Oct 1972 Henbane mosaic 160 Govier D D C M 1 A A B 95 June 1971 Influenza 700 Friedewald amp Pickels J Exptl Med 79 301 1944 Lettuce necrotic yellow 950 Lily symptomless Maize dwarf mosaic 155 Harrison amp Crowley Virology 26 290 1965 Allen T C C M I A A B 96 Oct 1972 Bancroft et al Phytopathology 56 474 1966 Maize dwarf mosaic B 171 3 Brakke amp Langenberg unpublished data Maize rough dwarf 400 Lovisolo O C M I A A B 73 Oct 1971 Narcissus mosaic 114 Mowat W P C M I A A B 45 June 1971 Okra mosaic T 42 B 106 Givord L amp Koenig R C M I A A B 128 July 1974 Papaya mosaic 118 7 Hiebert Phytopathology 60 1295 1970 Parsnip mosaic 149 Murant A F C M 1 A A B 91 June 1972 Peanut mottle Peanut stunt Bock K R amp Kuhn C W C M L A A B 141 Oct 1975 Mink G I C M 1 A A B 92 June 1972 Pea enation mosaic Shepherd R M C M A A B 25 Oct 1970 Pea seed borne mosaic Hampton R O amp Mink G
47. s slowly and since the rate of change in optical density will increase with faster speeds recording inaccuracies and poorer apparent resolution may result Teledyne Isco recommends starting at the minimum flow rate range listed in Table 3 1 for the selected centrifuge tube To set the flow rate at 0 375 mL min 1 Ensure that 0 065 1 16 ID 0 194 OD silicone tubing 029 1351 06 is installed in the pump 2 Place the X1 X10 MAX switch in the X10 position 3 Set the CONTROL dial to 15 To set the flow rate at 1 50 mL min 1 Ensure that 0 065 1 16 ID 0 194 OD silicone tubing 029 1351 06 is installed in the pump 3 1 Density Gradient Fractionation Systems Section 3 Operation 3 0 mL min Centrifuge Tube Size 2 Place the X1 X10 MAX switch in the X10 position 3 Set the CONTROL dial to 60 To set the flow rate at 3 0 mL min 1 Ensure that 0 125 Y8 ID 0 25 OD silicone tubing 029 1304 02 is installed in the pump 2 Place the X1 X10 MAX switch in the X10 position 3 Set the CONTROL dial to 36 M Note Refer to Section 3 of the Tris pump manual for complete infor mation on setting Tris pump flow rates Table 3 1 Recommended Flow Rates Mfg Rotor Designation Nominal Flow Rate mL min 7 16 x 1 15 16 Beckman 7 16 x 2 3 8 Beckman SW 56 10 9 x 54 7 mm International SB 405 1 2x2 Beckman SW 39 50 65 50 1 1 2x 2 Beckman Quick Seal 342412
48. such as e Combining the PEAK WIDTH with THRESHOLD detection e Using time windows to divert the initial tubing volume to waste e Renaming the method Refer to the user manual for more information At this time you should install racks and tubes on the Retriever 500 fraction collector Refer to Section 2 of the Retriever 500 Fraction Collector R2 user manual After installing the racks and tubes adjust the height of the drop counter if necessary Refer to Operating Procedures in Section 3 of the fraction col lector user manual and program the following nominal method which assumes e Tubes will be advanced every 78 pump counts roughly 1 mL e a 16 inch length of FEP 0 0625 inch ID tubing installed between the optical unit and diverter valve tubing E e a Y16 inch ID tube is in the Tris Pump To program the fraction collector 1 Place the POWER switch in the ON 1 position 2 Push the MODE button to select EXTERNAL 3 Push the DISPLAY button to select FRACTION SIZE 4 Push the UP ARROW button to enter volume counts as determined by the ID of tubing in the Tris Pump With Ys inch ID tubing the pump sends about 22 counts for each milliliter with 16 inch tubing the pumps sends about 78 counts for each milliliter For this example enter 78 The fraction collector is ready for operation The tube piercer uses a septum on the piercing mechanism to secure the bottom of the centrifuge tube The top assembly uses a
49. tinued Virus Absorbance 1mg mL 1 cm at 260 nm Reference Tobacco mosaic 3 0 Zaitlin M amp Israel H C M A A B 151 Oct 1975 Tobacco necrosis 118 5 0 5 5 Kassanis C M I A A B 151 June 1970 Tobacco necrosis satellite 50 6 5 Kassanis B C M I A A B 15 June 1970 Tobacco rattle Short 155 243 Long 300 3 0 Harrison B C M A A B 12 June 1970 Tobacco ringspot T 53 M 91 B 126 B 10 0 Stace Smith R C M L A A B 17 June 1970 Tobacco streak 90 113 Fulton R W C M I A A B 44 June 1971 Tomato aspermy 98 100 Hollings amp Stone C M A A B 78 Oct 1971 Tomato bushy stunt Tomato ringspot Tomato spotted wilt 131 140 T 53 B 126 128 Martelli C M A A B 69 Oct 1971 Stace Smith R C M L A A B 18 June 1970 Best Adv Virus Res 13 68 1968 Black Brakke amp Vatten Virology 20 120 1963 Tomato blackring Harrison 8 Nixon Virology 12 104 117 1960 Turnip crinkle Turnip yellow mosaic T 53 54 B 116 117 T 0 96 B 9 6 Hollings amp Stone C M A A B 109 Oct 1972 C M 1 A A B 2 June 1970 Wheat streak mosaic 165 Brakke M K Virology 6 96 1958 Wheat streak mosaic American Wheat striate mosaic 900 900 Yamazaki amp Kaesberg Biochem Biophys Acta 51 9 1961 Sinha R C amp Behki R M C M I A A B 99 Oct
50. tiple cartons Carefully unpack the shipment and inspect the contents A CAUTION Do not lift the Foxy R1 fraction collector by the arm If there is any damage to the shipping carton or any components contact the shipping agent and Teledyne Isco or its authorized representative immediately AN WARNING If there is any evidence that the system has been damaged in shipping do not plug it into AC power Contact Teledyne Isco or its authorized representative for advice Compare the contents of the boxes with the enclosed packing slips If there are any shortages contact Teledyne Isco immedi ately The Density Gradient Fractionation system is composed of several instruments Before these instruments may be used as a system you must first assemble the instruments and perform preliminary checkout procedures The instructions for these steps are found in the individual instrument user manuals The following sections specify the minimum assembly and checkout steps that should be accomplished before proceeding with your system installation as described in the sections titled Installation of System with Foxy R1 Fraction Collector on page 4 or Installation of System with Retriever 500 Fraction Collector on page 8 Unless otherwise necessary to complete the instructions in the individual manuals electrical cables and plumbing connections should only be completed when specified in this manual Density Gradient Fractionation Systems
51. tor Tubing and Hardware Kits As supplied with the Tris pump and fraction collector Manuals Supplied for the fraction collector UA 6 detector and Tris pump The systems do not form gradients in centrifuge tubes Density Gradient Fractionation Systems Section 1 Introduction 1 2 Specifications Specifications for the system are presented in Table 1 2 Table 1 2 System Specifications Overall Dimensions Height Width 22 5 Depth 17 5 61 7 cm 57 2 cm 44 5 cm 24 3 Weight With Foxy R1 28 7 kg 63 25 lbs With Retriever 500 24 6 kg 54 25 Ibs Does not include tube piercer racks tubes and fluids Power Requirements A 100 10 VAC 4 9 amperes 120 12 VAC 4 9 amperes 230 23 VAC 2 4 amperes Line Frequency 50 or 60 Hz Ambient Temperature 20 to 40 C maximum temperature must be at least 10 C above the boiling point of the lowest boiling solvent Humidity when connected to power 95 relative humidity maximum at 20 to 40 C Flow Rate Range 0 1 to 25 mL min Pump Speed Accuracy System Pressure 5 of full speed 0 to 2 7 bar CE Conformity Specifications Pollution degree 2 Installation category II Maximum altitude 2000 meters a Refer to the individual component manuals for additional specifications b Refer to the CE Declaration of Conformity at the back of the individual component manuals for applicab
52. ts Table A 2 Density at 25 C of various solutions used for density gradients Table A 3 Density of aqueous sucrose solutions g mL Table A 4 Centrifugal force chart for rotors commonly used in density gradient centrifugation Table A 5 Sedimentation rates and ultraviolet absor bances of some representative viruses A 1 Density Gradient Fractionation System Appendix A Tables Table A 1 Viscosity of various salt solutions used for density gradients Relative viscosity for a molal concentration of 2 3 4 5 10 1 454 1 479 1 673 1 895 0 817 1 007 K acetate 18 1 125 1 248 1 515 1 817 2 172 K tartrate 18 1 183 a Values selected from International Critical Tables and Landolt Bornstein Zahlen und Funktionen aus Physik Chemie Geophysik und Technik 7 Ed b 1 74 at 1 5 molal A 2 Density Gradient Fractionation System Appendix A Tables Table A 2 Density at 25 C of various solutions used for density gradients Concentration wt 30 40 K acetate K citrate 1 066 K tartrate 1 066 glycerol 1 021 sucrose 1 0381 Metrizamide Y Fluorinert FC48 1 93 g mL at 25 C Values are from International Critical Tables Highest values given are not necessarily for saturated solutions b The density of CsCl solutions may be calculated from the formula wt 137 48 138 11 1 p4 for 30 60 solutions Data from J Vinograd and
53. ument s technical reference manual The lightning flash and arrowhead within the triangle is a warning sign alert ing you of dangerous voltage inside the product Symboles de s curit Ce symbole signale l existence d instructions importantes relatives au produit dans ce manuel A A Ce symbole signale la pr sence d un danger d lectocution Warnungen und Vorsichtshinweise Das Ausrufezeichen in Dreieck ist ein Warnzeichen das Sie darauf aufmerksam macht daB wichtige Anleitungen zu diesem Handbuch geh ren gt gt Advertencias y Precauciones Der gepfeilte Blitz im Dreieck ist ein Warnzeichen das Sei vor gef hrlichen Spannungen im Inneren des Produkts warnt Esta se al le advierte sobre la importancia de las instrucciones del manual que acompa an a este producto gt gt Esta se al alerta sobre la presencia de alto voltaje en el interior del producto Density Gradient Fractionation Systems Safety vi Density Gradient Fractionation Systems Table of Contents Section 1 Introduction 1 1 System Features its at a aba Adve LE an 1 1 1 2 Specifications se dis ae a Shaw ee es ut a A ole 1 4 1 3 About this Manual 1 4 1 4 For Additional Information 1 4 Section 2 Installation 2 1 Unpacking cc iia iA da boa a beaded VE eee 2 1 2 2 System Assemblys eon 2 pu A
54. umulation des vapeurs explosives Teledyne Isco recommande que vous installez ce systeme dans un environnement bien a r qui est concu pour les mati res hasardeuses Cet environnement devrait tre construit selon les r glements f d raux provinciaux et locaux Aussi il devrait se conformer au plan de votre organisation qui concerne les m saventures de l hygi ne ou de chimique En tout cas utilisez toujours de pratiques bonnes de la laboratoire et des proc dures standardes de la s ret This manual applies Hazard Severity Levels to the safety alerts These three levels are described in the sample alerts below A CAUTION Cautions identify a potential hazard which if not avoided may result in minor or moderate injury This category can also warn you of unsafe practices or conditions that may cause property damage Ay WARNING Warnings identify a potentially hazardous condition which if not avoided could result in death or serious injury N DANGER DANGER limited to the most extreme situations to identify an imminent hazard which if not avoided will result in death or serious injury Hazard Symbols Warnings and Cautions gt gt Density Gradient Fractionation Systems Safety The equipment and this manual use symbols used to warn of hazards The symbols are explained below Hazard Symbols The exclamation point within the triangle is a warning sign alerting you of important instructions in the instr

Density Gradient Fractionation Systems User Manual

Contents

Download Pdf Manuals

Related Search

Related Contents