Idaho Technology Inc. Kit No. HRLS-ASY-0001

Transcription

1 Idaho Technology Inc. Kit No. HRLS-SY-0001

2 Kit Contents Quantity Description 500 µl 2.5X Master Mix 1 Tube Primer Set (Dry) 1 Tube Primer Set (Dry) 60 µl Reference DN Wild Type 60 µl Reference DN Het 1.5 ml Reagent Grade Water 1.8 ml Mineral Oil 100 µl 10X LCGreen Plus 1 LightScanner and LightScanner 32 Start-up Kit User s Guide LightScanner and LightScanner 32 Start-up User s Guide HRLS-PRT-0001 Rev. 8 08/ Idaho Technology, Inc., ll rights reserved. Information in this document is subject to change without notice. No part of this document may be reproduced or transmitted in any form or by any means, electronic or mechanical, for any purpose, without the express written permission of Idaho Technology, Inc. LightScanner, R..P.I.D., and Hi-Res Melting are registered trademarks of Idaho Technology, Inc. ll other names of products and brands appearing in this manual are trademarks or registered trademarks of their respective owners. The purchase of this product includes a limited, nontransferable license, under specific claims of one or more U.S. patents as listed on Idaho Technology, Inc. s Web site ( and owned by the University of Utah Research Foundation and/or Idaho Technology, Inc., to use only the enclosed amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of such U.S. patent(s), other than for the amount of product contained herein. The purchase of this product includes a limited, non-transferable license under U.S. Patent No. 5,871,908 and all continuations and divisionals, and corresponding claims in patents and patent applications outside the United States, owned by Roche Diagnostics GmbH, for internal research use or for non in vitro diagnostics applications with authorized reagents with regard to Melting Curve nalysis. No right is conveyed, expressly, by implication or estoppel, under any other patent or patent claims owned by Roche Diagnostics GmbH, or by any other Party. NOTE: Complying with all applicable copyright laws is the responsibility of the user. ii Idaho Technology Inc.

3 bout the Start-up Kit This LightScanner and LightScanner 32 (LS32 ) Start-up Kit from Idaho Technology provides all of the reagents necessary to set up 96 samples to be used for Hi-Res Melting on the LightScanner or LS32 instrument. This kit allows new LightScanner and LS32 users to familiarize themselves with the operation of both the instrument and the analysis software. The kit can also be used as a tool for verifying proper LightScanner or LS32 operation. This kit contains primers and DN that allow PCR amplification of two fragments of the 5' regulatory region of the human hepatic lipase gene. The fragments differ in length; Primer Set amplifies a fragment of 176 base pairs while Primer Set amplifies a fragment of 301 base pairs. The PCR reactions are well optimized and robust enough to be used with most common laboratory thermocyclers. lso included in the kit is a ready-to-use PCR Master Mix containing LCGreen Plus, wild type and heterozygous reference DN, reagent grade water for primer resuspension, and mineral oil (REQUIRED as an overlay for LightScanner operation). Equipment needed but not provided in kit: White-well, black-shell 96-well PCR plate (LightScanner) or sample carousel (LS32)* Glass capillaries (LS32) Sealing film (LightScanner)* Pipettes (1 10 µl, 200 µl) Plate centrifuge (LightScanner) Thermocycler *See ppendix for recommended PCR plates and sealing film. Idaho Technology Inc. 1

4 LightScanner Workflow Step 1: Wet Reaction Setup Follow basic PCR reaction setup using the Master Mix, primers and DN. The Master Mix contains LCGreen Plus. Master Mix Primers DN Step 2: Place Mix in Plate with Oil Overlay Preload PCR plate with mineral oil, add wet mix, seal with film and centrifuge briefly at 2500 rpm. Sealing Film Oil Overlay Step 3: Thermocycle Samples Follow the recommended protocols to perform PCR. PCR Step 4: Melt Samples in LightScanner Following PCR, insert the 96- or 384-well plate into a LightScanner to melt the samples. Step 5: nalyze Melt Data Following the melt, use the LightScanner software to manage and analyze the data. pproximately 5 to 8 Minutes Step 6: Scanning is Nondestructive Samples can be recovered for additional analysis, sequencing, gel electrophoresis, remelting, etc. Gel TCG Sequencing LSCN-PRT-0011 Rev 02 2 Idaho Technology Inc.

5 LightScanner Reaction Composition Primer Reconstitution 1. efore beginning, reconstitute Primer Sets and using reagent grade water as follows: dd 60 µl of water to the tube containing Primer Set dd 60 µl of water to the tube containing Primer Set The Primer Sets will now be at 10X concentration. 2. Thaw the frozen master mix and DN solutions on ice or at room temperature. Mix completely before using. Once thawed, reagents can be stored at 4 C for up to 2 weeks. Reaction Setup The following protocol results in the generation of four separate mixes. Each mix will have enough reagents to perform 24 reactions (including dead volume for pipetting error). The mixes will have the following composition ( = wild type; = heterozygous): Mix 1: Primer Set + DN Mix 2: Primer Set + DN Mix 3: Primer Set + DN Mix 4: Primer Set + DN Label four microcentrifuge tubes with the names indicated above. Idaho Technology Inc. 3

6 1. Primer Set and DN. Component Vol. (µl) Final Concentration 2.5X Master Mix 112 1X 10X Primer Set 28 1X Water 112 Template DN () ng/µl Final Volume Primer Set and DN. Component Vol. (µl) Final Concentration 2.5X Master Mix 112 1X 10X Primer Set 28 1X Water 112 Template DN () ng/µl Final Volume Primer Set and DN. Component Vol. (µl) Final Concentration 2.5X Master Mix 112 1X 10X Primer Set 28 1X Water 112 Template DN () ng/µl Final Volume Primer Set and DN. Component Vol. (µl) Final Concentration 2.5X Master Mix 112 1X 10X Primer Set 28 1X Water 112 Template DN () ng/µl Final Volume 280 Mix the reagent gently before dispensing (e.g., pipette up and down a few times). 4 Idaho Technology Inc.

7 Plate Setup Each reaction well should contain 10 µl of reaction mix and 15 µl of mineral oil overlay. Use the plate map below to distribute reagents C D E F G H Load the plate as follows: 1. liquot 15 µl of mineral oil into each well. 2. liquot 10 µl of reagents into the appropriate well. 3. Cover plate with sealing film. 4. Centrifuge 1 2 min. at rpm. lock Thermocycling Conditions Temp. ( C) Time (sec.) Step 1 Cycle Initial Denaturation Step 40 Cycles Denaturation nnealing/extension Heteroduplex Formation Idaho Technology Inc. 5

8 LightScanner Run Protocol 1. Follow the instructions in Chapter 4 of the LightScanner Operator s Manual, Running a LightScanner Experiment, to perform the melting protocol on the instrument. 2. Enter the following values in the Start, End, and Hold Temperature settings. LightScanner Melting Settings Start Temperature 75 C End Temperature 94 C Hold Temperature 65 C Exposure uto 3. Once the values have been entered, follow the directions in the Operator s Manual to load the plate into the instrument and start the run. LightScanner nalysis Protocol 1. Open the data file. Follow the instructions in Chapter 5, Common Functions for ll nalyses, of the LightScanner Operator s Manual to open the data file containing the results of the start-up kit melting protocol that you just ran. 2. Create two distinct analysis subsets for each primer set. The melting profiles for each primer set must be analyzed separately for best results. riefly, in the Subsets screen, select New Subset, select columns 1 6 of data, and select dd Marked to Subset. Name the subset Primer Set. For Primer Set, repeat by selecting New Subset and selecting columns 7 12 and dd Marked to Subset. Name the subset Primer Set. 3. Use the Expert Scanning module to group the data. Move into the Scanning Tab and choose the Primer Set subset, the raw melting curve data will be displayed on the graph. Under Standards choose uto Group, under Sensitivity choose Normal and hit Compute Groups. Change the graph 6 Idaho Technology Inc.

9 display to Temperature Shifted Data. Your samples should fall into two distinct groups based on their melting profiles. nalysis Protocol Example n example of the expected results for this start-up kit can be found in the LightScanner Software under: C:\LightScannerData\Scanning DemoData\96-well_InstallKit_data 1. To see this data, open the LightScanner Software, open the data file (.mat) listed above. The data file will display the plate map and the results as raw melting curves. 2. To see the Temperature shifted curves for Primer Set select the Primer Set analysis subset and the Temperature Shifted graph display from the pull down menu. 3. To see the Temperature shifted curves for Primer Set select the Primer Set analysis subset and the Temperature Shifted graph view from the pull down menu. Each nalysis subset will show two distinct groups of melting curves representing homoduplexes and heteroduplexes as illustrated below: Primer Set LightScanner Start-up Kit Primer Set Grouping Idaho Technology Inc. 7

10 Primer Set LightScanner Start-up Kit Primer Set Grouping If you do not get the expected result, please consult Chapter 10, The LightScanner Troubleshooting Guide, in the LightScanner Operator s Manual, or contact Idaho Technology Customer Support at support@idahotech.com. 8 Idaho Technology Inc.

11 LightScanner 32 Workflow Step 1: Wet Reaction Setup Follow basic PCR reaction setup using the Master Mix, primers and DN. The Master Mix contains LCGreen Plus. Master Mix Primers DN Step 2: dd reaction mix to capillaries. liquot 10 μl of reagents into the capillaries. Cap capillary. Spin capillaries to transfer reaction volume to bottom of tube. Step 3: Thermocycle Samples Follow the recommended protocols to perform PCR. PCR Step 4: Melt Samples in LightScanner 32 Following PCR, the LightScanner 32 will automatically perform a melt on the samples. Step 5: nalyze Melt Data Following the melt, use the LightScanner 32 software to manage and analyze the data. Step 6: Scanning is Nondestructive Samples can be recovered for additional analysis, sequencing, gel electrophoresis, remelting, etc. Gel TCG Sequencing Idaho Technology Inc. 9

12 LightScanner 32 Reaction Composition Primer Reconstitution 1. efore beginning, reconstitute Primer Sets and using reagent grade water as follows: dd 60 µl of water to the tube containing Primer Set dd 60 µl of water to the tube containing Primer Set The Primer Sets will now be at 10X concentration. 2. Thaw the frozen master mix and DN solutions on ice or at room temperature. Mix completely before using. Once thawed, reagents can be stored at 4 C for up to 2 weeks. Reaction Setup The following protocol results in the generation of four separate mixes. Each mix will have enough reagents to perform 9 reactions (including dead volume for pipetting error). The mixes will have the following composition ( = wild type; = heterozygous): Mix 1: Primer Set + DN Mix 2: Primer Set + DN Mix 3: Primer Set + DN Mix 4: Primer Set + DN Label four microcentrifuge tubes with the names indicated above. 1. Primer Set and DN. Component Vol. (µl) Final Concentration 2.5X Master Mix 36 1X 10X Primer Set 9 1X Water 36 Template DN () 9 10 ng/µl Final Volume Idaho Technology Inc.

13 2. Primer Set and DN. Component Vol. (µl) Final Concentration 2.5X Master Mix 36 1X 10X Primer Set 9 1X Water 36 Template DN () 9 10 ng/µl Final Volume Primer Set and DN. Component Vol. (µl) Final Concentration 2.5X Master Mix 36 1X 10X Primer Set 9 1X Water 36 Template DN () 9 10 ng/µl Final Volume Primer Set and DN. Component Vol. (µl) Final Concentration 2.5X Master Mix 36 1X 10X Primer Set 9 1X Water 36 Template DN () 9 10 ng/µl Final Volume 90 Mix the reagent gently before dispensing (e.g., pipette up and down a few times). Run Setup Each reaction capillary should contain 10 µl of reaction mix. Use the rotor map that displays on the software to distribute reagents (example below). Idaho Technology Inc. 11

14 1 8 Set Set Het 9 16 Set Het Set Het Load the capillaries and rotor as follows: 1. liquot 10 µl of reagents into the appropriate capillary. No oil! 3. Cap capillaries. 4. Spin capillaries to transfer reaction volume to bottom of tube. Program Denature Step Initial denaturation step PCR Conditions Temp. ( C) Time (sec.) cquisition Ramp rate ( C/sec) Denaturation Cycles mplification nneal Extension Single/ quanitifcation Pre-melt Heteroduplex formation and cooling chamber None Melt Hi-Res Melt 80 None 94 Continuous/ Hi-Res Melt 0.3 C 1 12 Idaho Technology Inc.

15 LightScanner 32 nalysis Protocol 1. Open the data file. Follow the instructions in Chapter 5, nalyzing bsolute Quantification Results, of the LightScanner 32 Operator s Manual, to open the data file containing the results of the startup kit melting protocol that you just ran. 2. Create two distinct analysis subsets for each primer set. Note: To select capillary locations for a subset, place the cursor so that it is hovering over the inner part of the carousel image (near the numbered slots). Run the cursor around the ring to highlight the desired cursor slots. The melting profiles for each primer set must be analyzed separately for best results. riefly, in the Subset Editor screen, click, select columns 1 16 of data, and select dd Marked to Subset. Name the subset Primer Set. For Primer Set, repeat by clicking, selecting columns 9 16, and selecting dd Marked to Subset. Name the subset Primer Set. Click pply. 3. Move into the Scanning Tab and choose the Primer Set subset, the raw melting curve data will be displayed on the graph. Under Standards choose uto Group, under Sensitivity choose Normal and hit Compute Groups. Change the graph display to Temperature Shifted Data. Your samples should fall into two distinct groups based on their melting profiles. nalysis Protocol Example n example of the expected results for this start-up kit can be found in the LightScanner 32 Software under: C:\LightScannerData\Scanning DemoData\96-well_InstallKit_data 1. To see this data, open the LightScanner Software, open the data file (.mat) listed above. The data file will display the plate map and the results as raw melting curves. 2. To see the Temperature shifted curves for Primer Set select the Primer Set analysis subset and the Temperature Shifted graph display from the pull down menu. Idaho Technology Inc. 13

16 3. To see the Temperature shifted curves for Primer Set select the Primer Set analysis subset and the Temperature Shifted graph view from the pull down menu. Each nalysis subset will show two distinct groups of melting curves representing homoduplexes and heteroduplexes as illustrated below: Primer Set LS32 Start-up Kit Primer Set Grouping 14 Idaho Technology Inc.

17 Primer Set LS32 Start-up Kit Primer Set Grouping If you do not get the expected result, please consult Chapter 10 in the LightScanner Operator s Manual or in the LightScanner 32 Operator's Manual, or contact Idaho Technology Customer Support at support@idahotech.com. Idaho Technology Inc. 15

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19 PPENDIX : Idaho Technology Master Mixes and Reagents Designed for Use with the LightScanner 96/384 and LightScanner 32 ssay LightScanner Master Mix 100 Reactions LightScanner Master Mix 500 Reactions High-Sensitivity Genotyping Master Mix 100 Reactions High-Sensitivity Genotyping Master Mix 500 Reactions Genotyping Master Mix 100 Reactions Warfarin Target Reactions Warfarin Target Reactions Warfarin Target Reactions Part Number HRLS-SY-0002 HRLS-SY-0003 HRLS-SY-0008 HRLS-SY-0009 HRLS-SY-0006 HRLS-SY-0007 HRLS-SY-0010 HRLS-SY-0011 The purchase of any of the above products includes a limited, nontransferable instrument license under specific claims of one or more U.S. patents as listed on Idaho Technology, Inc. s web site ( (the Web Site ) and owned by the University of Utah Research Foundation and/or Idaho Technology, Inc. ny kits (i) sold with this product and/or discussed herein may be covered by one or more of the U.S. patents, as listed on the Web Site for the product and (ii) sold herein include a limited, nontransferable license to use the enclosed amount(s) in such kits according to the specified protocols. HRLS-SY-0002, HRLS-SY-0003, HRLS-SY-0008, and HRLS-SY-0009 are licensed under U.S. Patent Nos. 5,338,671 and 5,587,287. The purchase of HRLS-SY-0002, HRLS-SY-0003, HRLS-SY-0006, HRLS-SY-0007, HRLS-SY-0008, HRLS- SY-0009, HRLS-SY-0010, and/or HRLS-SY-0011 includes a limited, non-transferable license for all fields other than human or veterinary in vitro diagnostics under U.S. Patent No. 5,871,908, owned by Evotec iosystems GmbH and licensed to Roche Diagnostics GmbH, to use only the enclosed amount of such products according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent No. 5,871,908, other than for the amount of product contained herein. Idaho Technology Inc. 17

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