Genotyping Automation Using Multiprobe II HT, Caliper AMS 90 SE, and Filemaker

1
Kaari Liisi Linask National Heart, Lung, and
Blood Institute
Genotyping Automation Using Multiprobe II HT,
Caliper AMS 90 SE, and Filemaker
7/22/2003
2
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

3
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 _at_ 55C with shaking for 4 hours on
  Thermomixer R with PCR plate adapter
• Heat inactivation of Proteinase K _at_ 95C for 10
  minutes on PTC 200 Thermocycler

4
Eppendorf Thermomixer R with 96-Well PCR Plate
Adapters
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Filemaker Genotyping Database

• Five linked files
• DNAList.fp5
• MasterMix.fp5
• Multiprobe.fp5
• PCRList.fp5
• 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.

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Required Sample Information from 7/8/2003 DNA
Plate

1. Sample or TagID
2. PlateID (Date)
3. Row (A-H)
4. Column (1-12)
5. Genotypes To Be Determined (Up to 3)

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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

8
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

9
DNAList.fp5 with samples from 7/8/2003 displayed
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Plate Numbering Conversions

• Multiprobe
• Numbered by column
• Well Row (8 X Column) - 8

• Caliper
• Numbered by row
• Well (12 X Row) Column -12

11
Generate Multiprobe Table Script

• Deletes previous sample records from the
  Multiprobe.fp5 file.
• Goes through each found records 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.

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Multiprobe.fp5 and MasterMix.fp5 Scripts

1. Export Multiprobe Source Data
2. Print Master Mix List
3. Export Caliper Source Data

13
Layout for Editing Master Mix Recipes
14
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

15
An example of samples 37 through 84 of a
Multiprobe.csv file exported from Filemaker and
opened in Excel

1. Master Mix Source
2. Master Mix Volume
3. Master Mix Tube
4. DNA Source Plate
5. DNA Volume
6. DNA Source Well
7. PCR Plate
8. PCR Well
9. of DNA Samples
10. PCR
11. DNA TagID

16
Caliper CSV Table Entry Guidelines
17
An example of the first 48 samples of a
Caliper.csv file exported from Filemaker and
opened in Excel

1. Concatenation of TagID and PCR
2. PCR (Master Mix)
3. Expected Fragment Size
4. Highlight Expected Fragment Flag

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MousePCR.MPT
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Multiprobe II HT Deck For Mouse Genotyping PCR
Set-Up
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Get DNA Sample Transfer Group

• Get 20 µL Tips
• Using info from source file
• Aspirate DNA
• Dispense DNA
• Drop 20 µL Tips
• Wash Tips

21
Close-up of Get DNA Sample Transfer Group
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Get Master Mix Transfer Group
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Tip Washing

• Wash tips used after each transfer group
• Wash all tips after transfer group node loop is
  completed

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PCR Cycling Conditions
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Caliper Electrophoresis

• Filter Gel-Dye Mixture
• 40 parts Gel Matrix
• 1 part Fluorescent Dye Concentrate
• Prepare DNA Ladder in PCR buffer
• 1X PCR Buffer
• 2mM MgCl2
• 1X DNA Ladder
• Prepare Wash Buffer
• 1X PCR Buffer
• 2mM MgCl2
• Prime Chip with Gel-Dye and add Markers
• Load Chip, Ladder, Wash Buffer, PCR Plate into
  the Caliper AMS 90 SE
• After running, rinse wells, and store chip with
  Storage Buffer
• 200mM TAPS
• 2mM EDTA
• pH 8.0 and 0.2µM filtered

26
Chip Diagram from the Caliper AMS 90 SE Users
Guide
27
Sample Plate, Ladder, and Wash Buffer Platform
from the Caliper AMS 90 SE Users Guide
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PCR Results

• Examples of results from a single plate of PCR.

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Row A Row B
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Row C Row D
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Row E Row F
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Row G Row H
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Example of a Single Lane Detail View
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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