User Manual: Color compensation Set (Dual color) (DIA-DAF(Lc2.0)-005) Ref.: Dia-DAF(Lc2.0)-005 DIAGENODE 27.08.2014 Color compensation Set (Dual Color) Fam Yellow Dye For Roche LightCycler® 2.0 www.diagenodediagnostics.com User Manual: Color compensation Set (Dual color) (DIA-DAF(Lc2.0)-005) DIAGENODE PAGE 3 Content Intended Use. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Fluorescent Dyes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Product Description. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Warnings & Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Required Materiel (not supplied). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Create a ccc object. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Performing a Color Hydrolysis probe Color compensation experiment 1. Capillaries preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2. PCR profile and procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Apply a ccc object. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Notice to Purchaser. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Explanation of Symbols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 www.diagenodediagnostics.com PAGE 4 DIAGENODE User Manual: Color compensation Set (Dual color) (DIA-DAF(Lc2.0)-005) Intended Use With this set, you can create an application-specific Color compensation object using Diagenode positive control from single color hydrolysis probe reactions. We recommend to perform Color compensation experiment once a year after the calibration of the optical part. Important notes • It is highly recommended to perform a new compensation file according to the master mix used. • The reagents in this Color compensation set have been elaborated especially to be performed with Diagenode's kits. Fluorescent Dyes • FAM (Max Abs 494 nm, Max Em 520 nm) • Yellow Dye (Max Abs 530 nm, Max Em 549 nm) Product Description 1. Dye adaptator file primers & double-dye probe: (FAM Dye) 90 µl, green tube. 2. Dye adaptator file primers & double-dye probe: (Yellow Dye) 90 μl, Yellow tube. 3. Positive control 4.103 copies/µl: 200 µl, Blank tube. 4. H2O (PCR grade): 1000 µl, white tube The product contains primers, probes and a positive control. The master mix is not included. Storage Color compensation set must be stored at -20°C until expiry date. STORE IN DARK PLACE. Diagenode sa / LIEGE SCIENCE PARK // Rue Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // Mail: [email protected] User Manual: Color compensation Set (Dual color) (DIA-DAF(Lc2.0)-005) DIAGENODE PAGE 5 Warnings and Precautions If, on receipt, a parcel is found to be damaged or if kits are thawed, please contact us : [email protected] / +32 4 364 20 52 Material Required (Not Provided) • Pipettes • PCR Lc2.0 Roche • Sterile pipette tips with filters (DNAse/RNAse free) • Capillaries tubes • Vortex • Laminar flow hood (Mix/DNA) www.diagenodediagnostics.com PAGE 6 DIAGENODE User Manual: Color compensation Set (Dual color) (DIA-DAF(Lc2.0)-005) Create a ccc object Performing a Color Hydrolysis probe Color compensation experiment You can create an application-specific Color compensation object using Diagenode positive control from single color hydrolysis probe reactions. 1. Capillaries Preparation Prepare 4 replicates Color compensation reactions for each fluorescence dye. 1)Mix Blank: Add the master mix and the Diagenode positive control, but without primers and probe and adjust volume with water. 2)Mix FAM: Prepare a PCR mix with Diagenode positive control, primers and a FAM-labeled hydrolysis probe for this control, the master mix, and adjust volume with water. 3)Mix Yellow: Prepare a PCR mix with Diagenode positive control, primers and a yellow dyelabeled hydrolysis probe for this control, the master mix, and adjust volume with water. We recommend to use with Lc 2.0, the LightCycler® taqman® Master Ref: 04535286001. PCR 20µl Blank FAM Yellow dye 4 µl 4 µl 4 µl - 2 µl 2 µl Diagenode Positive control 5 µl 5 µl 5 µl Water 11 µl 9 µl 9 µl Master Mix 5X Primers & double-dye probe Important notes : At the beginning of each run the LightCycler® instrument performs a seek process, which optimize positionning of the capillaries. 2. PCR profile and procedure 1) Select Run icon 2) In Programs, create your PCR program : Denaturation Step Cyles : 1 Target (°C) 95 Analysis Mode : none Hold (hh:mm:ss) 00:10:00 Slope (°C/s) Sec Target (°C) Step size (°C) Step Delay (cycles) Acquisition Mode 20 0 0 0 None Amplification Step Cycles : 45 Analysis Mode : None Target Hold Slope (°C) (hh:mm:ss) (°C/s) Sec Target (°C) Step size (°C) Step Delay (cycles) Acquisition Mode 95 00 :00 :15 20 0 0 0 None 60 00 :01 :00 20 0 0 0 None Diagenode sa / LIEGE SCIENCE PARK // Rue Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // Mail: [email protected] User Manual: Color compensation Set (Dual color) (DIA-DAF(Lc2.0)-005) DIAGENODE PAGE 7 Temperature Gradient Step Cycles : 1 Analysis Mode : Color Compensation Hold Target Slope (°C/s) Sec Target (°C) Step size (°C) Step Delay (cycles) Acquisition Mode 00:00:00 20 0 0 0 None 40 00:00:10 20 0 0 0 None 95 00:00:10 0.2 0 0 0 Continuous (°C) (hh :mm :ss) 95 Cooling Step Cycles : 1 Analysis Mode : none Hold Target (°C) (hh :mm :ss) 40 00:00:30 Slope (°C/s) Sec Target (°C) Step size (°C) Step Delay (cycles) Acquisition Mode 20 0 0 0 None NB : Setup : • Default Channel : 530 • Seek Temperature : 30 • Max. Seek Pos. : 3 • Instrument Type : 6 Ch. • Capillary Size : 20µl Sample Editing 1) Select Sample data icon 2) In Analysis Type & Capillary View, complete the table as follows: Pos Sample Name 1 Buffer 2 FAM 3 Yellow dye Repl. Of Sample Note NB : Sample Count : 3 Selected Channels : 530 3) In Analysis Type & Color Comp, complete the table as follows: Pos Sample Name Dominant Channel 1 Buffer Water 2 FAM 530 3 Yellow dye 560 www.diagenodediagnostics.com PAGE 8 DIAGENODE User Manual: Color compensation Set (Dual color) (DIA-DAF(Lc2.0)-005) Run 1)Place the reagent capillaries on the Carousel in the order listed above, making sure the blank sample (buffer) is always at position 1. 2)Start the run Analysis / Back up When the experiment is finished : 1) Select Color Compensation from the Analysis menu 2)Click Save cc object, then navigate to the folder Special Data\CCC under your user name and save the file as a ccc object in that folder. Apply a ccc object You can now apply the specific ccc object you create to any dual color Hydrolysis Probe experiment that is performed with FAM / Yellow dye combination, as follows : 1) Click Color compensation in the Run module or in the analysis window 2) Select Color Compensation 3)When a small dialog box opens and dispalys all compensated channels : 530 and 560, click OK The experimental data and/or the analysis graphs will now be redrawn using the compensated data. Notice to purchaser LightCycler® 2.0 instrument and LightCycler® taqman® Master are trademarks of Roche. Diagenode sa / LIEGE SCIENCE PARK // Rue Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // Mail: [email protected] User Manual: Color compensation Set (Dual color) (DIA-DAF(Lc2.0)-005) DIAGENODE PAGE 9 Explanation of symbols Catalogue number Batch code Number of experiments Temperature limitation Refer to user manual Positive control Manufacturer yyyy-mm Expiry Date Protect from the light www.diagenodediagnostics.com PAGE 10 DIAGENODE User Manual: Color compensation Set (Dual color) (DIA-DAF(Lc2.0)-005) Diagenode sa / LIEGE SCIENCE PARK // Rue Bois Saint-Jean, 3 // 4102 Seraing (Ougrée) // Belgium // Phone: (+32) 4 364 20 50 // Mail: [email protected] DIAGENODE PAGE 11 MA-DAF(Lc2.0)-V1_27_08_14 User Manual: Color compensation Set (Dual color) (DIA-DAF(Lc2.0)-005) www.diagenodediagnostics.com Diagenode sa LIEGE SCIENCE PARK Rue Bois Saint-Jean, 3 4102 Seraing - BELGIUM Tel. +32 4 364 20 50 [email protected] www.diagenodediagnostics.com
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