U.S. Department of Health and Human
Services
National Institutes of Health
National Heart, Lung, and Blood Institute
Genotyping Automation Using Multiprobe II HT, Caliper AMS 90 SE, and Filemaker
Genotyping Automation Using Multiprobe II HT, Caliper AMS 90 SE, and Filemaker
Kaari Liisi LinaskNational Heart, Lung, and Blood InstituteKaari Liisi LinaskNational Heart, Lung, and Blood Institute
7/22/20037/22/2003
Procedure Overview
1. Sample lysis
2. Filemaker generation of CSV data files for robotics software
3. Master mix preparation
4. Multiprobe PCR setup
5. Thermal-Cycling
6. Caliper electrophoresis
Tissue Lysis
• 2mm tail biopsies are lysed in a Nunc 96 well plate with 100µL lysis buffer containing 0.2 mg/mL Proteinase K
• Incubation @ 55°C with shaking for ~4 hours on Thermomixer R with PCR plate adapter
• Heat inactivation of Proteinase K @ 95°C for 10 minutes on PTC 200 Thermocycler
Eppendorf Thermomixer R with 96-Well PCR Plate Adapters
Filemaker Genotyping Database
Five linked files:1. DNAList.fp52. MasterMix.fp53. Multiprobe.fp54. PCRList.fp55. PostMix.fp5
Convert Required Sample info into a listing of PCRs in comma-separated value format (.csv) that the Multiprobe and Caliper software can access.
Required Sample Information from 7/8/2003 DNA Plate
G20 7/8/03 A 1 Cx43 TBDG21 7/8/03 B 1 Cx43 TBDG22 7/8/03 C 1 Cx43 TBDG23 7/8/03 D 1 Cx43 TBDG24 7/8/03 E 1 Cx43 TBDG25 7/8/03 F 1 Cx43 TBDG26 7/8/03 G 1 Cx43 TBDG27 7/8/03 H 1 Cx43 TBDG28 7/8/03 A 2 Cx43 TBDG29 7/8/03 B 2 Cx43 TBDG30 7/8/03 C 2 Cx43 TBDG31 7/8/03 D 2 Cx43 TBDG32 7/8/03 E 2 Cx43 TBDG33 7/8/03 F 2 Cx43 TBDG34 7/8/03 G 2 Cx43 TBDA22 7/8/03 A 3 ETA TBDA23 7/8/03 B 3 ETA TBDR843 7/8/03 A 4 R26R TBDR844 7/8/03 B 4 R26R TBDR845 7/8/03 C 4 R26R TBDR846 7/8/03 D 4 R26R TBDR847 7/8/03 E 4 R26R TBDR848 7/8/03 F 4 R26R TBDR849 7/8/03 G 4 R26R TBDM291 7/8/03 A 5 MHC43/DHFR TBD Test Cx43 TBDM292 7/8/03 B 5 MHC43/DHFR TBD Test Cx43 TBDM293 7/8/03 C 5 MHC43/DHFR TBD Test Cx43 TBDM294 7/8/03 D 5 MHC43/DHFR TBD Test Cx43 TBDM295 7/8/03 E 5 MHC43/DHFR TBD Test Cx43 TBDM296 7/8/03 F 5 MHC43/DHFR TBD Test Cx43 TBD
1. Sample or TagID
2. PlateID (Date)
3. Row (A-H)
4. Column (1-12)
5. Genotypes To Be
Determined (Up to 3)
DNAList.fp5 Scripts
1. Import DNA Plate Records
2. Find DNA Plate Records (up to 4)
3. Assign DNA Plate# (DNA1, DNA2, DNA3, DNA4)
4. Generate Multiprobe Table
DNAList.fp5 and PCRList.fp5 files• Converts the Row (A-H) and Column (1-12) to a
Well # with Multiprobe Numbering by column• Looks up and records the PCR required for each
PCR TBD based on the PCRList.fp5
DNAList.fp5 with samples from 7/8/2003 displayed
Plate Numbering Conversions
Multiprobe:
Numbered by column
Well# = Row + (8 X Column) - 8
Caliper:Numbered by rowWell# = (12 X Row) + Column -
12
Generate Multiprobe Table Script• Deletes previous sample records from the “Multiprobe.fp5” file.• Goes through each found record’s PCR fields (1A-3B). If there is
a reaction specified, a record is added to the “Multiprobe.fp5” file for each instance with Multiprobe well #, PCR master mix required, DNA TagID, Plate#, and PlateID recorded for each PCR reaction.
• Adds (-) controls for each PCR required.• Adds `H2O` wells to fill up the last partially utilized row of the
PCR plate (required for Caliper AMS 90 SE).• Finds, sorts, and numbers the needed PCR master mixes required
including `H2O` in the “MasterMix.fp5” file.• Sorts by PCR required and TagID (ascending ASCII)• Assigns PCR plate (currently up to 2), well row, and well column
numbers arranged for Caliper AMS 90 SE processing in rows.
Multiprobe.fp5 and MasterMix.fp5 Scripts
1. Export Multiprobe Source Data
2. Print Master Mix List
3. Export Caliper Source Data
Layout for
Editing Master
Mix Recipes
Print Master Mix List Script
• Format is in the same order as it will appear on the microfuge adapter plate
• Half of the plate fits on one page
An example of samples 37 through 84 of a Multiprobe.csv file exported from Filemaker and opened in Excel
A. Master Mix SourceB. Master Mix VolumeC. Master Mix Tube #D. DNA Source PlateE. DNA VolumeF. DNA Source Well #G. PCR PlateH. PCR Well #I. # of DNA SamplesJ. PCRK. DNA TagID
Caliper CSV Table Entry Guidelines
Column A Sample Name - Text only.Column B Sample Comments - Text only.Column C Expected Fragement Base Pair Size -
Numeric only (250;720;etc.).Multiple fragments can be entered.
Column D Highlight expected fragmentsfrom Column C on electropherogram?Yes = 1, No = 0 (Default is 0).
Cells A97+ Additional notes - Text only. Start entries in Column A.
An example of the first 48 samples of a Caliper.csv file exported from Filemaker and opened in Excel
A. Concatenation of TagID and PCR
B. PCR (Master Mix)C. Expected Fragment
SizeD. Highlight Expected
Fragment Flag
MousePCR.MPT
Multiprobe II HT Deck For Multiprobe II HT Deck For Mouse Genotyping PCR Set-UpMouse Genotyping PCR Set-Up
Get DNA Sample Transfer Group
QuickTime™ and aMotion JPEG OpenDML decompressorare needed to see this picture.
Source Plate DNA Volume DNA Well PCR Plate PCR WellDNA1 2 12 PCRPlate1 2DNA1 2 2 PCRPlate1 3DNA1 2 14 PCRPlate1 4DNA1 2 17 PCRPlate1 5DNA1 2 27 PCRPlate1 6DNA1 2 31 PCRPlate1 7DNA1 2 1 PCRPlate1 9DNA1 2 13 PCRPlate1 10
1. Get 20 µL Tips2. Using info from
source file:• Aspirate DNA• Dispense DNA
3. Drop 20 µL Tips4. Wash Tips
Close-up of Get DNA Sample Transfer Group
QuickTime™ and aMotion JPEG OpenDML decompressorare needed to see this picture.
Source Plate DNA Volume DNA Well PCR Plate PCR WellDNA1 2 7 PCRPlate1 57DNA1 2 36 PCRPlate1 58DNA1 2 9 PCRPlate1 59DNA1 2 38 PCRPlate1 60DNA1 2 37 PCRPlate1 61DNA1 2 26 PCRPlate1 62DNA1 2 8 PCRPlate1 65DNA1 2 37 PCRPlate1 66
Get Master Mix Transfer Group
QuickTime™ and aMotion JPEG OpenDML decompressorare needed to see this picture.
Source Volume Tube# PCRPlate PCR WellMasterMix 28 2 PCRPlate1 19MasterMix 28 2 PCRPlate1 20MasterMix 28 6 PCRPlate1 22MasterMix 28 1 PCRPlate1 25MasterMix 28 1 PCRPlate1 26MasterMix 28 2 PCRPlate1 27MasterMix 28 2 PCRPlate1 28MasterMix 28 5 PCRPlate1 29
Tip Washing
• Wash tips used after each transfer group
• Wash all tips after transfer group node loop is completed
QuickTime™ and aMotion JPEG OpenDML decompressorare needed to see this picture.
PCR Cycling Conditions
Hot-Start Program on PTC-200:1 95°C 5:00 minutes2 95°C 0:45 seconds3 58°C 0:30 seconds4 72°C 0.4°/second5 72°C 0:30 seconds6 Goto 2 39 times7 72°C 10:00 minutes8 4°C Forever9 End
Caliper Electrophoresis1. Filter Gel-Dye Mixture:
– 40 parts Gel Matrix– 1 part Fluorescent Dye Concentrate
2. Prepare DNA Ladder in PCR buffer:– 1X PCR Buffer– 2mM MgCl2
– 1X DNA Ladder3. Prepare Wash Buffer:
– 1X PCR Buffer– 2mM MgCl2
4. Prime Chip with Gel-Dye and add Markers5. Load Chip, Ladder, Wash Buffer, PCR Plate into the Caliper AMS
90 SE6. After running, rinse wells, and store chip with Storage Buffer:
– 200mM TAPS– 2mM EDTA– pH 8.0 and 0.2µM filtered
Chip Diagram from the Caliper AMS 90 SE User’s Guide
Sample Plate, Ladder, and Wash Buffer Platform from the Caliper
AMS 90 SE User’s Guide
PCR Results
• Examples of results from a single plate of PCR.
Row A Row B
Row C Row D
Row E Row F
Row G Row H
Example of a Single Lane Detail View
Adjustments and Capabilities That Have Been Added since
7/22/2003
• Adjustable master mix volumes
• Layout for addition of non-mouse database samples
• Multiple DNA source plates
• Multiple PCR plates
• Post PCR reagent addition