Nucleic Acid Quantitation (Nanodrop)[1]

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 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”

http://www.nanodrop.com/pdf/nd-1000-users-manual.pdf

http://www.nanodrop.com/pdf/nd-1000-users-manual.pdf

WADB STANDARD OPERATING PROCEDURE Number 008 NUCLEIC ACID QUANTITATION BY NANODROP® SPECTROPHOTOMETRY ______________________________________________________




(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)





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


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

of 10


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

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).





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.

http://www.nanodrop.com/





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





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:……………………………………………………...

Nucleic Acid Quantitation (Nanodrop)[1]

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