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 Kaari Liisi Linask National Heart, Lung, and Blood Institute Kaari Liisi Linask National Heart, Lung, and Blood Institute 7/22/2003

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 55°C with shaking for ~4 hours on Thermomixer R with PCR plate adapter Heat inactivation of Proteinase 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.fp5 2.MasterMix.fp5 3.Multiprobe.fp5 4.PCRList.fp5 5.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 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 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 `H 2 O` 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 `H 2 O` 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 Source B.Master Mix Volume C.Master Mix Tube # D.DNA Source Plate E.DNA Volume F.DNA Source Well # G.PCR Plate H.PCR Well # I.# of DNA Samples J.PCR K.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 A.Concatenation of TagID and PCR B.PCR (Master Mix) C.Expected Fragment Size D.Highlight Expected Fragment Flag

MousePCR.MPT

Multiprobe II HT Deck For Mouse Genotyping PCR Set-Up

Get DNA Sample Transfer Group 1.Get 20 µL Tips 2.Using info from source file: Aspirate DNA Dispense DNA 3.Drop 20 µL Tips 4.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 1.Filter Gel-Dye Mixture: –40 parts Gel Matrix –1 part Fluorescent Dye Concentrate 2.Prepare DNA Ladder in PCR buffer: –1X PCR Buffer –2mM MgCl 2 –1X DNA Ladder 3.Prepare Wash Buffer: –1X PCR Buffer –2mM MgCl 2 4.Prime Chip with Gel-Dye and add Markers 5.Load Chip, Ladder, Wash Buffer, PCR Plate into the Caliper AMS 90 SE 6.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