FISH Pre-Hyb Processing of Blood and Bone Marrow Cells
Contents
1. 085 USA 408 733 7337 techserv scigene com2. 300 ml 2 Protease Solution Table 1 Little Dipper Protocol for 0 01 NHCL 300 ml Pre Hybridization FISH Processing FISHBM1 Trypsin 0 15 g Temp Volume Agitation Time 5 Pepsin 240 ul step Bath Reagent cy tml step sec Add to Bath 2 when slides are in Bath 1 2x SSC 73 3 1xPBS 300 ml 4 1 Formaldehyde 1xPBS 250 ml Protease Solution 37 5 70 85 100 EtOH 250 ml each iene Instrument Setup ee 1 Rinse the removable baths stir bars and the processing 5 3 1x PBS RT 210 150 300 racks with 100 ethanol then with de ionized water three 70 EtOH E deville E 2 Place clean baths into positions 1 through 5 on the unit User pause Replace baths 3 4 with EtOH solutions Rotate all temperature sensors down 85 EtOH RT Note Any sensor remaining in the up position will 100 EtOH RT 510 50 60 interfere with the movement of the Little Dipper arm Continued on next page SciGene 617 N Mary Avenue Sunnyvale California 94085 USA 408 733 7337 techserv scigene com FISH Pre Hybridization Processing of Blood Cells and Bone Marrow Load Slides Run Protocol 1 Remove covers from Baths 1 and 2 2 Place slides ina 12 position rack for the Little Dipper instrument 3 Start the FISHBM1 protocol previously programmed Table 1 and load the rack containing the slides on the gripper as described in the Little Dipper Processor Operations Guide 4 Add the 2
3. 40 ul of 5 Pepsin to Bath 2 while slides are being processed in Bath 1 5 Turn off power to Baths 1 and 2 after steps 1 and 2 Table 1 are completed PML RARa DC DF 6 After completion of Step 5 the Little Dipper will pause and activate a beeping user alarm Replace baths in positions 3 4 with the EtOH solutions as indicated in Table 1 7 Resume protocol The instrument will complete the protocol through Step 8 Table 1 slowly withdrawing the rack presenting the slides for hybridization 8 Dispose of buffers and reagents at the end of the work day Wash baths and processing racks with warm water and rinse three times with de ionized water and dry with lint free towels Do not use detergents to clean baths Store baths and racks in a dust free environment ready for next use CLL CEP End Protocol CEP12 13q14 3 13q34 CEP4 CEP10 CEP17 FISH images of bone marrow cells using multiple probes Slides were processed following Day 1 pre hybridization and Day 2 post hybridization protocols on the Little Dipper Processor for FISH The Day 1 protocol is described here and the Day 2 protocol is described separately in a SciGene Method document for post hybridization of FISH assays using Vysis Probes Images compliments of Dr Teresa Smolarek Director of Cytogenetics Cincinnati Children s Hospital Medical Center SciGene Automating Array Workflows SciGene 617 N Mary Avenue Sunnyvale California 94
4. Little Dpper Processor for AH 63 gt Pub 120 Rev D 0609 FISH Pre Hybridization Processing of Blood and D il Bone Marrow Cells all Slide rack 12 position a we Bath 1 70 EtOH FH CH 2x SSC 210 ml RT 70 EtOH 85 EtOH 100 EtOH L ___ 210 mi RT 270 ml 73 C Bath 4 Bath 2 o D a Te a ml 37 Bath 3 1x PBS 210 ml RT 210 ml RT Equipment Configuration Instrument Setup continued e Little Dipper Processor for FISH 115v 230v 3 Using the touch screen create a protocol named SciGene cat 1080 70 1 1080 70 2 FISHBM1 or similar and enter the bath agitation rates and times and programmed pause as shown in Table 1 Consult the Little Dipper User Manual for details on creating and editing protocols e 2x Low volume temperature controlled baths SciGene cat 1080 10 5 for Baths 1 amp 2 e 2x Bath cover SciGene cat 1080 12 0 for Baths 1 amp 2 4 Fill each bath to the fill line with buffers shown in Table 1 e 6x Low volume baths and place covers over Baths 1 and 2 SciGene cat 1080 10 2 5 Turn on main power to the instrument and individual power i 12 positi i ides switch to Baths 1 and 2 only Set temperature on Bath 1 aaa aaa ineieliges to 73 C and Bath 2 to 37 C Wait 10 minutes for l temperature to stabilize Buffer and Reagent Preparation 6 Activate stir bars to all baths Set stir bar speed to 7 or just below maximum 1 2xSSC
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