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Guidelines

Guidelines for Sample Prep and QC

DNA Quantification Requirements for genotyping on the Illumina platform
The only method Illumina recommends for DNA quantification is PicoGreen quantification, using a spectroflurometer. We have had excellent results with DNA normalized after quantification using a NanoDrop spectrophotometer and quality controlled with agarose gel electrophoresis but please be advised that we cannot guarantee high quality data from DNA quantified by any other methods than those recommended by Illumina. We strongly discourage the use of plate reader spectrophotometers.

Note: When requested we will measure concentrations and OD260/OD280 of your samples in our Nanodrop spectrophotometer. When needed and requested we will concentrate samples by vacuum centrifugation. These are the only methods we can offer and we do not guarantee successful results when samples are not delivered at the requested concentration and volumes following Illumina’s guidelines (see above). An additional Service Fee will apply and these procedures will require one extra day in addition to the regular length of the corresponding Illumina protocol.

Illumina’s recommendations and Standard Operating Procedure for DNA quantification using PicoGreen:“Illumina’s genotyping method does not utilize an initial amplification of the genomic DNA. Therefore, the process is more sensitive to variation in DNA concentration than methods that do initially amplify the genomic DNA.”

PicoGreen® DNA Quantification SOP

1.0 REFERENCES
1.1 PicoGreen® dsDNA Quantification Reagent and Kits. Molecular Probes P7581
2.0 MATERIALS / EQUIPMENT REQUIRED
2.1 Disposables
2.1.1 96 well-plates as defined by customer’s equipment
2.1.2 Aluminum adhesive seals
2.1.3 50 mL serological pipette
2.1.4 50 mL conical tube
2.1.5 Aluminum foil
2.1.6 Multichannel pipette trough
2.2 Reagents
2.2.1 PicoGreen® dsDNA quantification reagent (Molecular Probes Cat # P7581)
2.2.2 1x TE (10mMTris/1mM EDTA)
2.2.3 Invitrogen Lambda DNA (Cat# 25250-028)
2.3 Equipment
2.3.1 Vortexer
2.3.2 Spectrofluorometer specific for PicoGreen®
2.3.3 Centrifuge
2.3.4 Mulitichannel Pipettor
3.0 PROCEDURE
3.1 Set up
3.1.1 Prepare lambda DNA Standards
3.1.1.1 Transfer 233.3 μL of 75 ng/μL lambda DNA to well A1 of a 96 well plate.(Fig 1).
3.1.1.2 Transfer 66.7 ul of 1X TE to well B of column 1 of the same 96-well 0.65 MIDI plate (Fig 1).
3.1.1.3 Transfer 100 ul of 1X TE to wells C, D, E, F, G, and H of column 1 of the same 96-well 0.65 mL MIDI plate (Fig 1).
3.1.1.4 Serially dilute (Fig 1) lambda DNA by transferring 133.3 μL of lambda DNA from well A1 into well B1. Pipette mix contents of well B1 five times, then transfer 100 μL from well B1 into well C1. Pipette mix contents of well C1 five times, then transfer 100 μL from well C1 into well D1. Pipette mix contents of well D1 five times, then transfer 100μL from well D1 into well E1. Pipette mix contents of well E1 five times, then transfer 100 μL from well E1 into well F1. Pipette mix contents of well F1 five times, then transfer 100 μL from well F1 into well G1.
Pipette mix contents of well G1 five times. Do not transfer solution from well G1 to well H1. Well H1 serves as the blank (0 ng/μL DNA).

3.1.1.5 The concentration of lambda DNA standards is given in Table 1.

3.1.1.6 Securely seal the Lambda standards plate with cap, label as “Lambda DNA Standard”, and store at 4°C for future use.
3.1.2 Prepare PicoGreen® Spectrofluorometer Plates
Caution: PicoGreen® is sensitive to photodegradation.
3.1.2.1 Remove PicoGreen® reagent from freezer and thaw at room temperature for 60 minutes in a light impermeable container.
3.1.2.2 Wrap aluminum foil around a 50 mL conical tube to prevent light penetration.
3.1.2.3 Make a 1:200 dilution of PicoGreen® to 1x TE in the 50 mL conical tube.

Dilutions will be made for a maximum of 2 sample plates at a time. Table 2
outlines the volumes needed of each reagent.

3.1.2.4 Cap the 50 mL dilution tube and mix with vortexer.
3.1.2.5 Pour PicoGreen® dilution into a clean multichannel pipette trough.
3.1.2.6 Using a multichannel pipette, transfer 195 μL PicoGreen® dilution to all 96 wells of the spectrofluorometer plate(s) (Fig 2). Immediately cover plate(s) with aluminum adhesive seal. This is the “DNA Sample Quant Plate”.
3.1.2.7 Repeat steps 6.1.2.3 to 6.1.2.6 so that there is one DNA Sample Quant plate for each DNA plate to be assayed.
3.1.2.8 Using a multichannel pipette, transfer 195 μL PicoGreen® dilution to rows A to H of columns 1 and 2 of a new 96 well spectrofluorometer plate (Fig 2). Immediately cover plate with aluminum adhesive seal and label “DNA Standard Quant Plate.”

3.1.3 Dilute DNA in PicoGreen®
3.1.3.1 These dilutions assume a sample DNA concentration between 0 and 50 ng/μL. Prepare your DNA accordingly before adding to the PicoGreen®. This protocol is quite accurate for final concentrations between 0 and 50ng/μL. Thus, if you plan on making a subsequent dilution for samples that quant between 50ng/μL and 75ng/μL using this protocol, we recommend that you dilute conservatively and recheck your final concentration using PicoGreen®. Using a multichannel pipettor transfer 2 μL of the Lambda DNA Standard to the DNA Standard Plate made in steps 3.1.2.8.
3.1.3.2 Mix contents of “Lambda DNA Standard” plates into the “DNA Standard Quant Plate” with a multichannel pipettor by pipetting up and down with at least 150 μL of the volume. Change tips between columns.
3.1.3.3 Using a multichannel pipet transfer 2 μL of each DNA to be assayed into the DNA Sample Plate(s) made in steps 3.1.2.6 to 3.1.2.7.
3.1.3.4 Mix contents of DNA Sample Quant Plate(s) with a multichannel pipet by pipetting up and down with at least 150 μL of the volume. Change tips between columns.
3.2 Measuring Fluorescence
Depends upon equipment available. Consult manufacturer’s recommendations.

RNA Requirements:
Please contact the facility for guidelines and current protocols.