sg series.qxp
Contents
1. an appropriate volume of a denser displacement solution to the mixing chamber and pump it through until all of the gradient mix has been delivered It is convenient to include a dye in the displacement solution to visually track the boundary between the gradient mix and the displacement solution e p3 sg series e generating convex and concave exponential gradients a 2 Generating convex and concave exponential gradients If the volume in the mixing chamber is held constant as opposed to declining for linear gradients the gradient generated will have an expo nential concentration curve The mixing volume in the SG 50 and SG 100 units can be held constant by sealing the top of the mixing chamber with the exponential gradient plunger accessory The total volume of the gradi ent can be greater than the maximum volume of the gradient former because additional solution can be repeatedly added to the reservoir chamber Using Eq 1 examples of the gradients generated by various combinations of volumes and concentrations listed in Table 2 are plotted Cr Vis A in Fig 3 Concave gradients are generated high concentration first and be is thus containers such as gel cassettes must be filled from the top Convex o V Noy gradients must be filled from the bottom Most frequently used is the res m x Vres concave pore gradient for increased resolution in the high molecular Eq 1 weight region of acrylamide gels To generate an exponential gr2. exponential plunger sg series e generating linear gradients um 80 6457 72 e p2 1 Generating linear gradients To generate a linear gradient between two concentrations equal volumes of solutions of the two concentrations are measured into the two chambers of the gradient former As solution is delivered out of the mixing chamber an equal volume flows in from the reservoir chamber where it is rapidly diluted and mixed to uniformity The initial concentration delivered will be that of the solution in the mixing chamber the final concentration will be that of the reservoir chamber For the most consistent delivery of gradients a peristaltic pump is recommended Table 1 Operating ranges for linear gradients Model max total vol ml min vol chamber ml max stir bar length mm SG 15 15 3 12 SG 30 30 5 15 SG 50 50 8 15 SG 100 100 16 20 1 Make sure all parts are clean and liquid flows freely through all channels stop cocks and tubing 2 Add a magnetic stir bar of the appropriate dimensions to the mixing chamber Table 1 and place the unit on a magnetic stirrer If volumes will be less than half the capacity of the unit an identical stir bar should be placed in the reser voir chamber as well to balance the displacement and prevent backflow into the reservoir when the chambers are first connected Connect tubing to the outlet connector and pump and adjust pump speed if used Position or connect the tubing to th
3. servicing or return unless this form is properly completed Hoefer is a trademark of Amersham Biosciences Limited or its subsidiaries Amersham and Amersham Biosciences is a trademark of Amersham plc O 2000 Amersham Biosciences Inc All rights reserved All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them A copy of these terms and conditions is available on request Printed in the USA Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany www amershambiosciences com Amersham Biosciences
4. use manual vertical electrophoresis SG Series SG 15 SG 30 SG 50 and SG 100 Gradient Makers Amersham um 80 6457 72 Rev BO 04 00 Biosciences Page finder Introduction Generating linear gradients Generating convex and concave exponential gradients Care and maintenance Ordering information Technical service and repair Important repacking instructions um 80 6457 72 pii plunger vent plunger body i plunger o ring e sg series instructions SG series instructions SG 15 SG 30 SG 50 and SG 100 gradient makers The Hoefer SG series gradient formers are designed for producing linear gradients of aqueous solutions ranging in volume from 15 100 ml The SG 50 and SG 100 units can be used to form convex and concave exponential gradients with the addition of an exponential gradient plunger accessory The small units SG 15 SG 30 SG 50 and SG 100 are machined from solid blocks of acrylic plastic They are suitable for casting acrylamide pore gradient gels casting immobilized pH gradient gels pouring density gradients for centrifugal sedimentation separations and delivering salt gradients for low pressure chromatography systems reservoir back chamber connector channel mixing front chamber stopcock washer delivery stopcock outlet connector 4 mm id tubing Fig 1 SG series gradient former and
5. adient 1 Make sure all parts are clean and liquid flows freely through all channels stop cocks and tubing 2 Attach to the red plunger vent a short piece of flexible vinyl tubing that can be clamped off to make an airtight seal 3 Add a magnetic stir bar of the appropriate dimensions to the mixing chamber and place the unit on a magnetic stirrer Connect tubing to the outlet connec tor and pump then adjust pump speed if needed Position or connect the tubing to the receiving vessel gel casting unit centrifuge tube etc at top or bottom as appropriate 4 Close both stopcocks handle up and add a portion of the final solution to the reservoir back chamber 5 Carefully open the connector stopcock and allow just enough solution to flow through the connector channel to fill it to the edge of the mixing chamber then close the stopcock Be sure no large bubbles remain to obstruct flow through the channel 6 Add the required volume of the starting solution to the mixing chamber With the plunger vent open insert the plunger into the mixing chamber and press it down until it is just above the surface of the solution Fig 2 Clamp off the tubing on the vent airtight N Start the magnetic stirrer and open the delivery stopcock 8 Simultaneously open the connector stopcock and start the pump Note if the gradient volume is larger than the reservoir will hold the addi tional volume can be added during the gradien
6. e receiving vessel gel casting unit centrifuge tube etc 3 Close both stopcocks handles up and add the required volume of the final solution to the reservoir back chamber 4 Carefully open the connector stopcock and allow just enough solution to flow through the connector channel to fill it to the edge of the mixing chamber then close the stopcock Be sure no large bubbles remain to obstruct flow through the channel 5 Add the required volume of the starting solution to the mixing chamber and start the magnetic stirrer 6 Open the delivery stopcock 7 Simultaneously open the connector stopcock and start the pump Note if there is a substantial difference in densities between the two solutions there will be a sudden flow from the denser chamber to the lighter chamber to bring the two into hydrostatic balance This will result in the gradient not being fully linear To avoid this add equal weights rather than volumes of the solu tions to the appropriate chambers 8 If it is important that no bubbles disturb the gradient watch the delivery care fully and as soon as the last of the solution has entered the pump head stop the pump and remove the tubing from the receiving container 9 Flush and rinse all parts thoroughly with distilled water after use e pouring gradients 1 1 Pouring gradients from the top Filling a container with a gradient e g casting acrylamide pore gradient gels can be done either dense solu
7. ent maker should be cleaned thoroughly with distilled water after use to prevent polymerization or crystallization of solutions in the chambers and stopcocks Do not use abrasives acetone pure alcohols or organic solvents to clean this unit 4 Ordering information Qty Code No SG 15 Gradient Maker 15 ml total volume 1 80 6197 61 SG 30 Gradient Maker 30 ml total volume 1 80 6197 80 SG 50 Gradient Maker 50 ml total volume 1 80 6197 99 SG 100 Gradient Maker 100 ml total volume 1 80 6196 09 Plunger for exponential gradient SG 50 1 80 6198 75 Plunger for exponential gradient SG 100 1 80 6197 23 White 3 mm outlet fitting SG 15 SG 30 SG 50 SG100 1 80 6196 85 Black Luer fitting SG 15 SG 30 SG 50 1 80 6196 66 Stopcock standard size for SG 15 SG 30 SG 50 1 80 6196 28 Long stopcock for SG 100 connector stopcock 1 80 6197 04 Short stopcock for SG 100 delivery stopcock 1 80 6196 47 SG 500 Gradient Maker 500 ml total volume 1 80 6198 18 5 Technical service and repair Amersham Biosciences offers complete technical support for all our products If you have any questions about how to use this product or would like to arrange to repair it please call or fax your local Amer sham Biosciences representative 6 Important repacking instructions Request a copy of the Amersham Biosciences Contamination Clearance Certificate before returning the item No items can be accepted for
8. t delivery Do not allow the reservoir chamber to go dry as bubbles will be pulled into the mixing chamber changing the dilution volume and gradient shape Fig 2 Gradient maker with plunger in place 9 Stop the pump and remove the tubing from the receiving container as soon as the last of the solution has left the reservoir chamber Note The entire solution remaining in the mixing chamber is at the final concentration and does not contribute further to the gradient If desired it can be used as a displacement solution for convex gradients unless it contains acrylamide 10 Disconnect tubing Flush and rinse all parts thoroughly with distilled water after use um 80 6457 72 p4 e examples of concave and convex exponential gradients Table 2 Examples of concave and convex exponential gradients A B c D E Cmix 1 0 0 0 5 1 Vmix 20 20 50 20 20 Cres 0 1 1 1 0 5 Vres 100 100 10 100 100 c concentration v volume mix mixing chamber res reservoir chamber 1 0 0 9 A 0 8 I B 0 7 S 06 i E 5 0 5 o g 0 4 4 0 3 A 0 2 0 1 C 0 0 T T T T 1 0 20 40 60 80 100 of gradient delivered Fig 3 Examples of exponential gradients sg series e care and maintenance um 80 6457 72 e p6 3 Care and maintenance The gradi
9. tion first from the top or light solution first from the bottom To fill from the top 1 Proceed as described above putting light final or top solution in the reservoir chamber step 3 and dense solution in the mixing chamber step 5 2 Place the delivery outlet against the upper edge of the receiving container Adjust the pump rate so that the solution flows evenly down the side in a smooth continuous stream The delivery speed should be slow enough that the newly arriving solution does not mix with the underlying solution Alternatively using a rigid cannula at the end of the delivery tubing hold the tip of the cannula just above the surface of the solution raising it smoothly as the container fills 1 2 Pouring gradients from the bottom This technique is commonly used in filling multiple gel casting chambers 1 Proceed as described above putting dense final or bottom solution in the reservoir chamber step 3 and light solution in the mixing chamber step 5 2 Connect the delivery tubing to the bottom inlet of a gel casting unit or to a cannula long enough to reach the bottom of the receiving container Adjust the pump rate so that the solution is not forced up in a fountain that mixes with the overlying solution 3 If all of the gradient solution must be delivered to the container a displace ment solution may be used Just as the last of the gradient mix is pumped out of the mixing chamber add
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