Hoefer PR648
Contents
1. Setting up the Hoefer PR648 0 Place the bottom block on the lab bench Place the membrane support block middle block with the O ring facing down on top of the bottom block The membrane recess in the membrane support block has one curved corner Orient the block with this corner on the lower right see Fig 1 o Wear gloves to avoid leaving fingerprints on the membrane Place the membrane cutting template ridge side down on the membrane Exert slight pressure on the template to leave a mark on the membrane Cut the membrane along the mark The template has been sized to allow for swelling of all types of membrane e Soak the membrane in appropriate buffer for a few minutes to wet it thoroughly A dry membrane will draw sample under the sealing ridge and away from the slot resulting in a poorly defined slot image on the membrane o Place the membrane completely within the recess aligning the clipped corner of the membrane with the clipped corner of the recess Make sure the membrane is flat and fits completely within the area with edges parallel to the recess edges If the membrane is not positioned correctly lift it up and reposition it Note Do not over tighten Over tightening causes leaks Fig 2 Correct order for tightening the screws Fig 3 Correct set up of the liquid trap e Carefully set the top block with letters and numbers facing up on top of the membrane supp2. solutions can be used Do not use ethanol or other organic solvents Do not autoclave the slot blot or wash it at high temperatures e Blot all blots dry immediately Acrylic can absorb water which may cause it to warp o Store the slot blot away from direct sunlight If you store it assembled do not tighten the screws e Store the PR648 slot blot with the membrane cutting template flat side up between the middle and top blocks This will protect the sealing ridge surrounding the slots Troubleshooting problem solution If the sample area on the membrane is larger than the slot size or if the solution is leaking out of the slot Screws are tightened too far or not tightened far enough The membrane is not positioned correctly with the recess The membrane is dry Carefully follow the tightening instructions in Steps 6 and 7 in Setting up the Hoefer PR648 slot blot Make sure the membrane is flat and fits completely within the recess with its edges parallel to the recess edges Before blotting be sure to soak the membrane in buffer to wet it thoroughly If small impressions show on the membrane around the blot This is normal If there are bubbles in the slot There are proteins or detergent in the sample Or the sample was squirted rapidly into the slot The impressions which indicate a tight seal will not affect your results if you scan the membrane in reflec
3. eo manual blotting Hoefer PR648 Slot blot manifold i 1 jt f e Ades me um PR648 IM Rev F0 08 12 Ato e fe r 9 Page finder L ouireTe rode e 1 SPECHICAL ONS iae ee eur a trav e o ena 2 Procedure for standard use of the Hoefer PR648 essssssseeeee mee 3 Setting up the Hoefer PR648 Applying your samples aee cene Removing your DIOE esee secessit ri 5 Care and malint marce ea cicius tento er eri seen 6 Troubleshoobltig iioii ore pepe Dar atop AR o ar P4baX Rhe sc 9 Ordering information esee rette tonio eut sata 8 Introduction Slot and dot blotting techniques have been used extensively in molecular biology to immobilize nucleic acids and proteins on membranes for determining nucleic acid homologies quantify ing mRNA studying hormone receptor binding detecting protein nucleic acid interactions and screening for specific proteins by activity or antibody The slot shaped blots produced by the Hoefer PR648 slot blot filtration manifold are more reliably and accurately quantitated by scanning densitometry than dot blots For a series of dots the scan path must pass directly through the center of all the dots Slots are not so demanding in alignment and allow much wider representative scan paths The Hoefer PR648 slot blot filtration manifold is designed and manufactured to provide an efficient seal around each slot insuring a consis tent shape and size As little a
4. ize is necessary to distribute the sample evenly over the slot To prevent bubbles from forming pipet each sample against the bottom side wall of the well This is especially important it your sample contains proteins or detergents If bubbles should form flush them out with the pipetter tip o Turn on the vacuum and set it at 13 25 cm Hg When all of the sample liquid has been pulled through the membrane add 1 ml of buffer to each slot pipetting against the bottom side wall Adjust vacuum to 38 50 cm Hg After all of the buffer is pulled through repeat twice more for a total of three rinses Removing your blot 0 With the vacuum still on remove the screws and carefully lift off the top block o Using forceps lift the membrane off and place it on clean dry filter paper Turn off the vacuum e Process the membrane according to your protocol o If you plan to scan the blot itself with a densitometer dry the membrane flat by placing it on the smooth side of a porous polyethulene sheet Dry it for several minutes in a vacuum gel dryer without heat Care and maintenance 0 If the slot blot is used with radioactive materials decontaminate the O ring between uses You may wish to purchase a second ring for non radioactive work See Ordering information o Wash all blocks thoroughly in a mild water based laboratory detergent then rinse well in distilled water Brief exposure to 5 bleach
5. ort block o Insert the screw into the threaded holes Tighten the screws until the knobs just touch the surface of the block 9 Hand tighten each screw only a small amount following the sequence shown in Fig 2 Repeat the sequence one or two times until all screws are snug e Attach the slot blot to a small vacuum pump with adjustable vacuum and a vacuum gauge If an adjust able vacuum pump is not available use a water aspi rator or a house vacuum and place a bleed valve and vacuum gauge in the vacuum line To keep liquids from being sucked into the vacuum source set up a liquid trap between the slot blot and the vacuum source as shown in Fig 3 a Use a side arm vacuum flask and a stopper with a small hole b Insert a glass tube through the stopper hole Position the tube so that it extends below the flask sidearm when the stopper is in place c Stopper the flask Use a length of rubber tubing to connect the glass tube in the stopper to the connector on the Hoefer PR648 d Use a second length of tubing to connect the side arm of the flask to the intake port of the vacuum pump or to the vacuum line Note Do not use samples containing organic solvents Applying your samples 0 Turn on the vacuum pump and adjust the pump or bleed valve until the vacuum is 13 25 cm Hg o Turn off the vacuum e Carefully load a sample at least 50 ul in volume into each well A 50 ul s
6. s 50 pl of sample applied to a membrane through the slot will yield a clearly defined band with evenly distrib uted sample Each Hoefer PR648 slot blot is comprised of three separate blocks The upper block has 48 labeled slots for samples arranged in a 4 x 12 array The blotting membrane is placed in a recess on the top of the middle membrane support block The bottom block has a connec tor for the vacuum source To assemble the Hoefer PR648 slot blot simply place the membrane in the membrane support block recess stack the blocks together and insert and tighten the screws Attach the connec tor on the bottom block to a vacuum source and apply your samples in the slots Specifications Number of slots Slot dimensions Slot spacing Slot membrane surface area Maximum well volume Recess dimension Unit dimensions Fig 1 The Hoefer PR648 slot blot set up for standard use 48 6 mm x 0 80 mm 9 0 mm x 18 mm from center to center 4 8 mm 1 ml 82 mm x 115 mm 12cm x 15 5 cm x 8 cm top block membrane membrane recess membrane support block orientation corner quick disconnect bottom block Procedure for standard use of the Hoefer PR648 The following procedure produces a well defined blot with sharp edges with many types of samples To determine the optimal load per slot for the particular membrane and samples you are using we suggest first blotting a set of serial dilutions
7. tance mode Place the membrane flat on the smooth side of a porous polyethylene sheet and dry it for several minutes in a gel dryer without heat To prevent bubbles from forming be sure to pipet the sample slowly and carefully against the bottom side wall of the slot If necessary flush bubbles out with a pipette tip Ordering information product qty code no Basic Unit Hoefer PR648 Slot blot manifold 1 PR648 with Quick Fit connector and membrane cutting template Replacement Parts for the PR648 Slot blot Top Block PR654 Membrane support block PR659 middle block Includes O ring Bottom Block PR656 O rings 1 PR657 Screws 6 PR658 Membrane cutting template 1 PR659 Hoefer Inc 84 October Hill Road Holliston MA 01746 Toll Free 1 800 227 4750 Phone 1 508 893 8999 Fax 1 508 893 0176 E mail support hoeferinc com Web www hoeferinc com Hoefer is a registered trademark of Hoefer Inc 2012 Hoefer Inc All rights reserved Printed in the USA Hoefer
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