GE RPN2512E manuale d’uso

GE Healthcare Amersham Hybridization oven/shak er Pr oduct User Manual Codes: RPN2510E RPN2511E RPN2512E[...]

RPN2510EUM Pagefinder Rev C 06/2007 2 Page finder 1. Legal 3 2. Introduction 4 3. Safety warnings and precautions 5 3.1. Electrical Installation 5 4. Specification 6 5. Setting up the Hybridization O ven/Shak er 7 5.1. Setting the oven temperature 7 5.2. Setting up the rotisserie 7 5.3. Setting up the platform shaker 8 6. Hybridization using the pl[...]

1. Legal GE, imagination at work and GE monogram are trademarks of General Electric Company. Amersham, Alkphos Direct , ECF , ECL , ECL Plus, ECL Advance, Hybond, Hypercassette, Hyperfilm, Hyperscreen, Hyper torch, Megaprime, Nick, Rapid- hyb, Ready-To-Go , Rediprime, Sensitive and Trackertape are trademarks of GE Healthcare companies. All third pa[...]

2. Intr oduction One of the most widely used techniques in the molecular biolog y field is the immobilization of DNA and RNA onto a solid suppor t membrane and subsequent hybridizations with a specific single stranded probe, labeled to facilitate its detection. Using the GE Healthcar e Hybridization O ven/Shaker ensures that the temperature and sha[...]

3. Safety w arnings and pr ecautions If the equipment is not used in the manner described in this manual the protection provided might be impaired This equipment is designed to operate under the following conditions: - For indoor use only Use in a well ventilated area Ambient temperature range + 5°C to + 40°C Altitude to 2000 m Relative humidity [...]

4. Specification Overall dimensions Height: 17” (43.5 cm) Depth: 15” (38.0 cm) Width: 15” (38.0 cm) Oven dimensions Height: 8” (20.0 cm) Depth: 9” (23.0 cm) Width: 10” (25.0 cm) Weight: 24 kg Capacity (nominal): 18 litres T emperature range: Ambient plus 5°C–80°C T emperature precision: +/- 0.5°C T emperature f luctuation: +/- 0.1?[...]

5. Setting up the Hybridization O ven/Shaker RPN2510EUM Chapter 5 Rev C 06/2007 7 Fig 1. Diagram of instrument control panel Rotisserie/shaker controls 1. Rotisserie/shaker ON/OFF button 2. Rotisserie/shaker speed selector dial 3. Motor on indicator Temperatur e controls 4. Temperature on/off button 5. Press to set button 6. LED temperature display[...]

4. Place the rotisserie into the oven onto the rotation mechanism, ensuring that the serrated bands at either end of the rotisserie locate onto the steel cogs of the rotation mechanism at the rear of the oven. (B on figure 2). The plastic flanges of the r otisserie locate on to the small wheels on the oven floor . Close the oven door . 5. Ensure th[...]

RPN2510EUM Chapter 6 Rev C 06/2007 9 6. Hybridization using the platform shaker The Hybridization Oven/Shaker is compatible with the hybridization technologies available from GE Healthcare. These include radioactive hybridizations using Rapid hyb buffer and the range of non-radioactive systems for the labelling and detection of proteins and nucleic[...]

RPN2510EUM Chapter 7 Rev C 06/2007 10 Several advantages are associated with performing hybridizations in bottles, namely those of safety and economy as outlined in the introduction. However , the use of bottles for hybridizations and washing procedures requires certain adaptations to standard protocols. 7.1. Assembly of membranes into bottles 1. A[...]

RPN2510EUM Chapter 7 Rev C 06/2007 11 4. Prehybridize the membrane for the specified length of time at a rotisserie speed of 4 rpm. 5. Following prehybridization, turn off the rotisserie, remove the rotisserie from the oven. Add the labeled probe to the buffer , either by removing an aliquot of the buffer , adding the probe and returning the aliquo[...]

RPN2510EUM Chapter 8 Rev C 06/2007 12 8. Maintenance/car e/cleaning of the oven/shak er The GE Healthcare Hybridization Oven/Shaker is designed to provide trouble-free operation. The base of the oven and the shaker tray act as a spills tray and will contain any spillage that occurs during hybridization and washing procedur es. T o ensure lasting op[...]

RPN2510EUM Chapter 9 Rev C 06/2007 13 Symptom 1. Membrane curling up in hybridization bottle 2. High background 3. Weak signal 4. Pat chy backgrounds 5. High background around edge of membrane Cause 1.1. Incorrect orientation of bottle in rotisserie 2.1. Probe concentration too high 2.2. Unincorporated 32 P nucleotides not removed 2.3. Insufficient[...]

Appendix 1. Pr oducts for electr ophor esis DNA Markers Precise Sizing Digest of Natural DNAs 50 Base-pair 100 Base-pair KiloBase DRigest III ladder ladder DNA marker 27-4060-01 27-4005-01 27-4007-01 27-4004-01 500 a 2000 a 10000 23130 b 450 1900 8000 9416 400 1800 6000 6557 350 1700 5000 4361 b 300 1600 4000 2322 250 1500 3000 2027 200 1400 2500 1[...]

Appendix 2. Hybond membranes for nucleic acid applications Size Pack size Hybond N+ Hybond XL Hybond N Hybond NX Hybond ECL Hybond C Extra Hybond P 82 mm 50 discs RPN82B RPN82S RPN82N RPN82T RPN82D RPN82E 87 mm 50 discs RPN87B RPN87S RPN87N RPN87T 132 mm 50 discs RPN132B RPN132S RPN132N RPN132T RPN132D 137 mm 50 discs RPN137B RPN137S RPN137N RPN137[...]

Appendix 3. Radioactive labeling systems Labelling T echnology Nucleotide Amount of Labelling Probe specific Recommended Product system template time activity application code (dpm/µg) Rediprime ™ II Random-prime dCTP only 25 ng 10 minutes 2 × 10 9 membrane RPN1633 hybridization RPN1634 Ready-T o-Go ™ Random-prime dCTP only 10 ng–1 µg 5 mi[...]

Appendix 4. Non-radioactive labeling and detection systems Labelling and detection Sensitivity Time from Duration of Strip before Quantification Recommended application system hybridization light output re-pr obing to detection AlkPhos Direct (RPN3690) 0.06 pg 1 hour 5 days yes no Single copy Southern and Northerns ECL Direct (RPN3000) 0.5 pg 1 hou[...]

Appendix 5. Pr oducts for autoradiography and chemiluminscent detection Hyperfilm – high performance autoradiography films Product description size number sheets code Hyperfilm ECL 5 × 7 inches 50 28-9068-35 Hyperfilm ECL 18 × 24 cm 50 28-9068-36 Hyperfilm ECL 18 × 24 cm 100 28-9068-37 Hyperfilm ECL 8 × 10 inches 50 28-9068-38 Hyperfilm ECL 8[...]

Appendix 6. Hybridization buffer Product name Pack size Code Rapid-Hyb buffer Rate enhanced hybridization buffer 125 ml RPN1635 for use with radiolabelled nucleic acid probes 500 m RPN1636 RPN2510EUM Appendix 6 Rev C 06/2007 19[...]

Appendix 7: Radiation safety pr oducts Product Description Product Dimensions Quantity Code Number CDC storage box 240 × 140 × 130 mm 1 box RPN2032 Pipette guards Beta pipette guard, Gilson P20/100 1 guard RPN1544 Beta pipette guard, Gilson P200 1 guard RPN1545 Beta pipette guard, Gilson P1000 1 guard RPN1546 Multipack, 1 each of the above codes [...]

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RPN2510EUM Rev C 06/2007 imagination at work GE Healthcare offices: GE Healthcare Bio-Sciences AB Björkgatan 30, 751 84 Uppsala, Sweden GE Healthcare Europe GmbH Munzinger Strasse 5, D-79111 Freibur g, Germany GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue, P .O . Box 1327, Piscataway, NJ 08855-1327, USA GE Healthcare Bio-Sciences KK Sanke[...]