# Presentation: 396425

## 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 Linask**
**National Heart, Lung, and Blood Institute**

**7/22/2003**

## Procedure Overview

- Sample lysis
- Filemaker generation of CSV data files for robotics software
- Master mix preparation
- Multiprobe PCR setup
- Thermal-Cycling
- 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:
- 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.

## Required Sample Information from 7/8/2003 DNA Plate

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

## DNAList.fp5 Scripts

- Import DNA Plate Records
- Find DNA Plate Records (up to 4)
- Assign DNA Plate# (DNA1, DNA2, DNA3, DNA4)
- 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 row
- Well# = (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

- Export Multiprobe Source Data
- Print Master Mix List
- 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

- Master Mix Source
- Master Mix Volume
- Master Mix Tube #
- DNA Source Plate
- DNA Volume
- DNA Source Well #
- PCR Plate
- PCR Well #
- # of DNA Samples
- PCR
- DNA TagID

## Caliper CSV Table Entry Guidelines

## An example of the first 48 samples of a Caliper.csv file exported from Filemaker and opened in Excel

- Concatenation of TagID and PCR
- PCR (Master Mix)
- Expected Fragment Size
- Highlight Expected Fragment Flag

## MousePCR.MPT

## Multiprobe II HT Deck For Mouse Genotyping PCR Set-Up

## Get DNA Sample Transfer Group

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

## Close-up of Get DNA Sample Transfer Group

## Get Master Mix Transfer Group

## Tip Washing

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

## PCR Cycling Conditions

## 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

## 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