nucleic acid quantitation (nanodrop)[1]
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STANDARD OPERATING PROCEDURE Number 008
NUCLEIC ACID QUANTITATION BY NANODROP® SPECTROPHOTOMETRY
______________________________________________________
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
I. PRINCIPLE
• The NanoDrop® ND-1000 UV-Vis Spectrophotometer enables highly accurate analyses of 1 μl
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/ μl, without dilution.
• Full-spectrum UV-Vis absorbance analyses (220-750nm)
• Sample is recoverable (except RNA)
II. REFERENCES
1. ND-1000 Spectrophotometer V3.3 User’s Manual (available at http://www.nanodrop.com/pdf/nd-1000-
users-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”
WADB STANDARD OPERATING PROCEDURE Number 008 NUCLEIC ACID QUANTITATION BY NANODROP® SPECTROPHOTOMETRY ______________________________________________________
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
(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)
WADB STANDARD OPERATING PROCEDURE Number 008 NUCLEIC ACID QUANTITATION BY NANODROP® SPECTROPHOTOMETRY ______________________________________________________
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
VI. GENERAL REAGENTS
(1) Milli-Q water MILLIPORE Milli-Q Reagent Water System
18MΩ 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. DEFINITIONS None
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 μl
volumes
WADB STANDARD OPERATING PROCEDURE Number 008 NUCLEIC ACID QUANTITATION BY NANODROP® SPECTROPHOTOMETRY ______________________________________________________
SOP 008 Nucleic Acid Quantitation by Nanodrop® Spectromphotometry
XII. PROCEDURE
Wear gloves and laboratory safety gear throughout procedure.
Set up the Computer i. Access the Desktop screen on computer attached to the Nanodrop® ND-1000
ii. Double click on the Nanodrop® icon (ND1000 V3.30) and the Main Menu screen will appear
iii. Double click on ‘User’ box and select ‘DNA Bank’ from drop down menu
iv. Enter WADB password
v. 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
vi. 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
vii. Select the “Nucleic Acid’ application module (When measuring RNA, change the drop down box to
‘RNA-40’)
viii. 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 User’s 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).
©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 NUCLEIC ACID QUANTITATION BY NANODROP® SPECTROPHOTOMETRY ______________________________________________________
SOP 008 Nucleic Acid Quantitation by Nanodrop® Spectromphotometry
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)
5. Exit the application by clicking on the “Exit” button (note the measurements will automatically save)
6. Log out of the User account by clicking “Exit” button on the main menu screen
7. Always keep the pedestal closed when not in use
8. Perform ‘Calibration Check’ every 6 months according to manufacturers instructions (see Section
XIII)
9. Arrange re-calibration by technician if Calibration Check falls outside acceptable parameters (as
advised by manufacturer) or fails.
Notes for Cleaning the Sample Retention System o Initial cleaning with 70% Ethanol followed by Milli Q water before any measurements are made
o 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
©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 NUCLEIC ACID QUANTITATION BY NANODROP® SPECTROPHOTOMETRY ______________________________________________________
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
o Although generally not necessary, 2 μl 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 user’s 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).
WADB STANDARD OPERATING PROCEDURE Number 008 NUCLEIC ACID QUANTITATION BY NANODROP® SPECTROPHOTOMETRY ______________________________________________________
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
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.
WADB STANDARD OPERATING PROCEDURE Number 008 NUCLEIC ACID QUANTITATION BY NANODROP® SPECTROPHOTOMETRY ______________________________________________________
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 ______________________________________________________
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
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 μg of DNA corresponds to an
absorbance of 1 at 260 nm.
DNA concentration (μg/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/bio-
mol-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
WADB STANDARD OPERATING PROCEDURE Number 008 NUCLEIC ACID QUANTITATION BY NANODROP® SPECTROPHOTOMETRY ______________________________________________________
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 10 of 10
NUCLEIC ACID QUANTITATION BY NANODROP® SPECTROPHOTOMETRY
DOCUMENT HISTORY
Date Issue Number Author Description of Amendment Authorised by
1/11/05 1 Erna Lin Transferred from MBM John Beilby
9/11/06 2 Erna Lin Annual Review John Beilby
1/12/06 3 Marion Macnish,
Simone Dowd &
Laura Greenwood
Modified for WADB Use
(incorporation of diagrams
and User Account details)
John Beilby
1/5/08 3.1 Marion Macnish,
Simone Dowd &
Laura Greenwood
Add requirement for
calibration check every 6
months and full instructions
John Beilby
Authorised by:…………………………………………..
(signature)
Date:……………………………………………………...