STANDARD OPERATING PROCEDURE Number 008

NUCLEIC ACID QUANTITATION BY NANODROP SPECTROPHOTOMETRY

______________________________________________________ I.

PRINCIPLE
The NanoDrop ND-1000 UV-Vis Spectrophotometer enables highly accurate analyses of 1 l samples (DNA, RNA, dyes, proteins and microbial cell culture) No cuvettes or capillaries are required Accurately and reproducibly measures nucleic acid samples up to 3700 ng/ l, without dilution. Full-spectrum UV-Vis absorbance analyses (220-750nm) Sample is recoverable (except RNA)

II.

REFERENCES

1. ND-1000 Spectrophotometer V3.3 Users Manual (available at http://www.nanodrop.com/pdf/nd-1000users-manual.pdf) (Also hard copy stored next to Nanodrop in Rm F50.8)

III.

SPECIMEN
Nucleic acids DNA (from any extraction method) RNA (from any extraction method)

IV. STOCK REAGENTS

All reagents are located as follows: a) room temperature (RT) stock reagents are kept on the shelves in Rm F50.8. b) room temperature (RT) working reagents are kept on the shelves in Rm F46.1. c) +4 oC reagents are kept in refrigerator in Rm. F46.1. d) -80 oC freezers are located in the passage between J and G blocks on the 1st floor. e) Autoclaving is done at 125 oC for 15 mins for plasticware and 134 oC for 5 mins for glassware and reagents. Autoclave is located in Rm. F45.4 (1st floor, J Block). Instructions for the handling of the autoclave is in the WADB SOP001 Autoclave Instructions

SOP 008 Nucleic Acid Quantitation by Nanodrop Spectromphotometry Western Australian DNA Bank Standard Operating Procedures Manual Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood Version 3.1 Effective date: 01/05/08 Page 1 of 10

WADB STANDARD OPERATING PROCEDURE Number 008

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(1) 1M Tris-HCl, pH 8.0 1.21 g Trizma base (SIGMA Cat. no. T1503) Dissolve in 800 ml Milli-Q water and adjust pH to 8.0 with 1 M Hydrochloric acid. Adjust final volume to 1.0 L. Autoclave buffer for 5 minutes to sterilise. Store at RT. Discard Tris solution if it is yellow in colour.

(2) 0.5M Ethylenediamine Tetra-acetic acid (EDTA) Solution pH 8.0 186.1 g of Disodium EDTA Powder (Sigma Aldrich, Cat. No. E5134-250G) 800 ml of Milli-Q water Adjust to pH 8.0 with 10M NaOH. Adjust final volume to 1.0 L.Store at 4o C

(3) 100% (Absolute) Ethanol Ethanol (BDH Analar Cat. No. 10106 4D) Store an aliquot (500 ml) at -20 oC

V.

WORKING REAGENTS

(1) TE Buffer, pH 8.0 (10 mM Tris-HCl, 1mM disodium EDTA) 10 ml 1M Tris-HCL, pH 8.0 2 ml 0.5M EDTA Adjust final volume to 1.0 L with Milli-Q water. Autoclave buffer for 5 minutes to sterilise. Store at RT

(2) 70% Ethanol Mix 350 ml ethanol with 150 ml Milli-Q water. Store at -20oC in the 70%EtOH bottle

(3) RNA BR5 buffer (for RNA quantitation only) (from Qiagen Paxgene Blood RNA kit see WADB SOP007 RNA Extraction using the Paxgene Blood RNA System for details)

(4) RNAse free water (for RNA quantitation only) (any source including from Qiagen Paxgene Blood RNA kit)

SOP 008 Nucleic Acid Quantitation by Nanodrop Spectromphotometry Western Australian DNA Bank Standard Operating Procedures Manual Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood Version 3.1 Effective date: 01/05/08 Page 2 of 10

WADB STANDARD OPERATING PROCEDURE Number 008

NUCLEIC ACID QUANTITATION BY NANODROP SPECTROPHOTOMETRY

______________________________________________________ VI. GENERAL REAGENTS

(1) Milli-Q water MILLIPORE Milli-Q Reagent Water System 18M water is available from the Milli-Q system located in room F45.4. Special Chemistry has responsibility for the system's maintenance and service.

VII. REAGENTS SUPPLIED BY MANUFACTURER

CF -1 Calibration Solution (Biolab Cat No NDT CF-1)

VIII. STANDARDS
None

IX. QUALITY CONTROL

None

X.
None

DEFINITIONS

XI. EQUIPMENT
NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., USA) (1 each in Laboratory F50.8 and Laboratory F43.3) Precision pipette (eg Gilson P2 pipette) and aerosol resistant filter pipette tips suitable for 1 -2 l volumes

SOP 008 Nucleic Acid Quantitation by Nanodrop Spectromphotometry Western Australian DNA Bank Standard Operating Procedures Manual Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood Version 3.1 Effective date: 01/05/08 Page 3 of 10

WADB STANDARD OPERATING PROCEDURE Number 008

NUCLEIC ACID QUANTITATION BY NANODROP SPECTROPHOTOMETRY

______________________________________________________ XII. PROCEDURE

Wear gloves and laboratory safety gear throughout procedure.

Set up the Computer i. ii. iii. iv. v. vi. vii. viii. Access the Desktop screen on computer attached to the Nanodrop ND-1000 Double click on the Nanodrop icon (ND1000 V3.30) and the Main Menu screen will appear Double click on User box and select DNA Bank from drop down menu Enter WADB password CLEAN the upper and lower measurement pedestal first with 70% ethanol then by Milli Q water on a lint-free wipe (eg Kimwipes) DO NOT USE TISSUES INITIALISE (pre prime) the instrument using 2 ul deionised water (if measuring DNA) or 2 ul RNAse free water (if measuring RNA) as outlined in steps 1 3 below. At step 2 click on OK button Select the Nucleic Acid application module (When measuring RNA, change the drop down box to RNA-40) BLANK (zero) the instrument as outlined in steps 1 - 3 below this must be done using the same diluent the DNA or RNA is suspended in (usually TE buffer if measuring DNA or BR5 buffer if measuring RNA extracted using the the Paxgene RNA system), At step 2 click on Blank button for Blank only (note; samples will thereafter be measured using the Measure button) ix. Before loading the sample enter the sample details into Sample ID box by scanning the 2D barcode with hand held scanner (DS6608-HD laser scanner, Symbol, USA) (or manually enter ID code). 1. With the sampling arm open (Figure 1a), pipette the sample onto the lower measurement pedestal (Figure 1b.(Images from Nanodrop ND-1000 Users Manual))

Figure 1a

Figure 1b

2. Close the sampling arm. The sample column is automatically drawn between the upper and lower measurement pedestals and the spectral measurement made (Figure 1c). Click on Measure box to initiate a reading (allow approx 10 secs for measurement).
SOP 008 Nucleic Acid Quantitation by Nanodrop Spectromphotometry Western Australian DNA Bank Standard Operating Procedures Manual Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood Version 3.1 Effective date: 01/05/08 Page 4 of 10

WADB STANDARD OPERATING PROCEDURE Number 008

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Figure 1c

3. The concentration of the sample will appear in the ng/ul column. Enter the concentration details directly into the appropriate study LIMS before moving on to the next sample measurement, When the measurement is complete open the sampling arm and wipe the sample from both the upper and lower pedestals using lint free wipes (eg Kimwipes) (Figure 1d). Simple wiping prevents sample carryover in successive measurements for samples varying by more than 1000 fold in concentration.

Figure 1d

4.

If a printed copy of the report is required, select the Print Report button (at any time the user can display the measurements and/or rename the Sample ID by selecting the Show Report button) Exit the application by clicking on the Exit button (note the measurements will automatically save) Log out of the User account by clicking Exit button on the main menu screen Always keep the pedestal closed when not in use Perform Calibration Check every 6 months according to manufacturers instructions (see Section XIII) Arrange re-calibration by technician if Calibration Check falls outside acceptable parameters (as advised by manufacturer) or fails.

5. 6. 7. 8.

9.

Notes for Cleaning the Sample Retention System o o Initial cleaning with 70% Ethanol followed by Milli Q water before any measurements are made Wiping the sample from both the upper and lower pedestals after each sample measurement is usually sufficient to prevent sample carryover and avoid residue buildup

SOP 008 Nucleic Acid Quantitation by Nanodrop Spectromphotometry Western Australian DNA Bank Standard Operating Procedures Manual Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood Version 3.1 Effective date: 01/05/08 Page 5 of 10

WADB STANDARD OPERATING PROCEDURE Number 008

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o Although generally not necessary, 2 l water aliquots can be used to clean the measurement surfaces after particularly high concentration samples to ensure no residual sample is retained on either pedestal o After measuring a large number of samples it is recommended that the areas around the upper and lower pedestals be cleaned thoroughly. This will prevent the wiping after each measurement from carrying previous samples onto the measurement pedestals and affecting low-level measurements o A final cleaning of all surfaces with 70% Ethanol followed by Milli Q water is also recommended after the users last measurement.

Decontamination of Measurement Pedestals o If decontamination is necessary, a sanitising solution, such as a 5.25% solution of sodium hypochlorite (bleach freshly prepared), can be used to ensure that no biologically active material is present on the measurement pedestals (the metal fiber optic fittings are made from 303 stainless steel and are resistant to most common laboratory solvents).

Sample Recovery o If required samples can be recovered from the upper and lower measurement pedestals by extraction with a pipette (not RNA as surface is not RNAse-free).

SOP 008 Nucleic Acid Quantitation by Nanodrop Spectromphotometry Western Australian DNA Bank Standard Operating Procedures Manual Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood Version 3.1 Effective date: 01/05/08 Page 6 of 10

WADB STANDARD OPERATING PROCEDURE Number 008

NUCLEIC ACID QUANTITATION BY NANODROP SPECTROPHOTOMETRY

______________________________________________________ XIII. ND-1000 CALIBRATION CHECK

Ref: adapted from 2006 Nanodrop Technologies Inc, USA

INSTALL SOFTWARE Download and install the latest version of the ND-1000 Calibration Check Software from the Support Section on the Nanodrop website at www.nanodrop.com

CALIBRATION PROCEDURE 1) Ensure the measurement pedestals are clean and that a 1 ul water sample beads up on the lower pedestal. 2) Open the ND-1000 Calibration Check Software and follow the prompts in the Customer Guidance text box of the software. i. Enter the Target Absorbance found on the CF-1 vial into text box (typically the target absorbance is 0.734: actual value will depend of the lot of CF-1) ii. Add 1ul of deionised water and select Blank 3) Before opening the glass ampoule of CF-1 Calibration Fluid shake vigorously to ensure solution is thoroughly mixed. Ensure all solution is collectied in the bottom portion of the ampoule. 4) Carefully break the neck of the glass ampoule to open the CF-1 Calibration Fluid (Care broken glass hazard) 5) Follow the on-screen prompts in the Customer Guidance text box. Using individual 1 ul samples of the CF-1 Calibration Fluid, measure 10 replicates. 6) After the 10th measurement the calibration check results will be displayed on-screen in the Customer Guidance text box. If the instrument does not pass the calibration check using 1 ul samples immediately rerun the procedure again (step 5) using 2 ul samples. 7) To print a copy of the results for your records clinck the Print Screen button. 8) If recalibation is required contact local Australian supplier (Biolab)

Note: the CF-1 Calibration Fluid is supplied in a single-use vial. The CF-1 must be used within one hour of opening the vial. Exposure to the environment or transferring of the fluid to another container may cause a significant concentration change.

SOP 008 Nucleic Acid Quantitation by Nanodrop Spectromphotometry Western Australian DNA Bank Standard Operating Procedures Manual Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood Version 3.1 Effective date: 01/05/08 Page 7 of 10

WADB STANDARD OPERATING PROCEDURE Number 008

NUCLEIC ACID QUANTITATION BY NANODROP SPECTROPHOTOMETRY

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SOP 008 Nucleic Acid Quantitation by Nanodrop Spectromphotometry Western Australian DNA Bank Standard Operating Procedures Manual Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood Version 3.1 Effective date: 01/05/08 Page 8 of 10

WADB STANDARD OPERATING PROCEDURE Number 008

NUCLEIC ACID QUANTITATION BY NANODROP SPECTROPHOTOMETRY

______________________________________________________ XIV. CALCULATIONS

None automated read out provided by spectrophotometer

Principle of calculations Concentration The estimation of DNA concentration is based on the observation that 50 g of DNA corresponds to an absorbance of 1 at 260 nm. DNA concentration (g/ml) = Absorbance260 x 50 x dilution factor (eg.200/10) Purity The ratio of the absorbance at 260 and 280 nm indicates the purity of the DNA. DNA purity = A260/A280 The ratio should be 1.8 - 2.0. A ratio of <1.8 may indicate the presence of phenol or protein contamination, unless DNA extracted from saliva in which case the ratio may be less due to excess turbidity of the sample prior to processing (>1.60). If the OD ratio is low (<1.8) can continue with Ethanol precipitation of DNA extracted by any protocol (WADB SOP003, SOP004, SOP005, SOP006).

XV. REPORTING
Copy of the DNA concentration of samples report is saved on PathWest server (Nt008crmpc/biochem/biomol-biology/WA DNA Bank) (note: A file titled Nucleic Acid 2005 02 08.ndt. corresponds to nucleic acid data from 8 Feb 2005. A unique file extension (.ndt) allows start-up with MS Excel).

XVI. PRECAUTIONS AND HAZARDS

None

XVII. DOCUMENT HISTORY

See Master Copy

SOP 008 Nucleic Acid Quantitation by Nanodrop Spectromphotometry Western Australian DNA Bank Standard Operating Procedures Manual Prepared by: Marion Macnish, Simone Dowd & Laura Greenwood Version 3.1 Effective date: 01/05/08 Page 9 of 10

WADB STANDARD OPERATING PROCEDURE Number 008

NUCLEIC ACID QUANTITATION BY NANODROP SPECTROPHOTOMETRY

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NUCLEIC ACID QUANTITATION BY NANODROP SPECTROPHOTOMETRY

DOCUMENT HISTORY
Date 1/11/05 9/11/06 1/12/06 Issue Number 1 2 3 Author Erna Lin Erna Lin Marion Macnish, Simone Dowd & Laura Greenwood 1/5/08 3.1 Marion Macnish, Simone Dowd & Laura Greenwood Description of Amendment Transferred from MBM Annual Review Modified for WADB Use (incorporation of diagrams and User Account details) Add requirement for calibration check every 6 months and full instructions John Beilby Authorised by John Beilby John Beilby John Beilby

Authorised by:.. (signature) Date:...

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