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National Center for Environmental HealthCenters for Disease Control and Prevention
Laura Hancock, M.S.Molecular Quality Improvement Program
Newborn Screening and Molecular Biology Branch,Division of Laboratory Sciences
NCEH, CDC
NBS Molecular Resources Website2016 NBS Molecular Training Workshop
NBS Molecular Resources WebsiteResources Available
Find liquid handling options by viewing detailed summaries of instruments used by other NBS programs
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Access the Molecular Assessment Program (MAP) site visit checklist and request a MAP visit
www.aphl.org
New Addit ion to Website – Pipett ing Verificat ion
New Addit ion to Website – Pipett ing Verificat ion
New Addit ion to Website – Pipett ing Verificat ion
Pipetting Verification
State CDCMethod Used Orange G and absorbance readingContact Name Laura Hancock
Contact Phone Number 770-488-4617Contact email [email protected]
Secondary Contact Name Suzanne CordovadoSecondary Contact Phone Number 770-488-4048
Secondary Contact email [email protected] SOP available upon request? Yes
Instrument(s) Model tested and pipetting mechanism Biomek FXp & Biomek Nxp
pipetting mechanismdrop down menu (can we select multiple options?):
- 96 channel- 8 channel
- single channel
96 channel and 8 channel
Range of Volumes that can be tested using this method All volumes used in on liquid handler are verified 1ul - 200ul
Are there any MSDS concerns with the chemicals/reagents listed? What concerns are noted? No
How long does the verification take? 1-3 hours, depending on difficulty of programmed method
Short Description of Method (2-3 sentences)
The instrument dispenses a solution of Orange G of the volume of interest into a 96-well plate and water is added. The absorbance is measured on a spectrophotometer at absorbance 492nm and each well’s volume is calculated based on a standard curve, as well as the plate average volume, standard deviations, %CV, and minimum and maximum volumes.
How often are instrument(s) calibrated? Biomek FXp - two times a yearBiomek Nxp - two times a year
Are pipetting volumes verified by an outside source, as well as verified by NBS lab? no
Can pipetting tool be removed from instrument for pipetting verification, or is it part of the instrument? no
Instrument(s) NeededMolecular Device's SpectraMax M2 absorbance readervortexmanual pipettors
Solutions NeededConcentrations of Orange G in water is based on the volume that is being verified. All absorbance readings should be between 0.8 - 1.3 on the spectrophotometer at A492.
How are volumes selected for verification? w/drop down menu options:
- All volumes used in on liquid handler are verified- min/max/mid point volumes used on liquid handler are
verified- only select critical volumes are verified
- blank (for free-form answer)
All volumes used in on liquid handler are verified
If select volumes are verified, what volumes?
1. A standard curve is created using calibrated manual pipettes and read on the absorbance reader at A492.2. Using Microsoft Excel, a line of best fit is determined from the standard curve.3. In a 96 well plate, the instrument dispenses the desired volume of Orange G and water is added to bring the total volume up to 100 ul.4. The Orange G/water plate is gently mixed on a vortex for approximately 20 seconds.5. The Orange G/water plate is read in the absorbance reader at A492.6. Using Microsoft Excel, the volume delivered into each well is calculated using the line of best fit and the absorbance reading.7. The average, the minimum, and the maximum volumes delivered, as well as the %CV are calculated
Detailed Description of Pipetting verification method
Volumes delivered by the instrument are determined acceptable by the end user based on the specific needs of the method the volume will be used in, as judged by average volume delivered, standard deviation, %CV, and minimum and maximum volumes delivered.
A %CV ≤5% is acceptable when dispensing 2µL and a %CV of ≤2% is acceptable when dispensing larger volumes.
The minimum and maximum acceptable range is determined based on volume dispensed and method(s) used.
Acceptable Results
Data generated is analyzed using Microsoft Excel. The standard curve is plotted on a scatter plot, and excel generates a trend line and the formula for that trend line. All absorbance readings are copied from the Spectrophotometer software and pasted into excel. The trend line equation is used to calculate the volume pipetted from the absorbance readings. The trend line equation is y=mx+b, where m (slope) and b (y-intercept) is calculated in excel based on the scatter plot, and y is the absorbance reading of the volume pipetted.
Analysis Software (how is pipetting data analyzed?)
A standard curve is generated immediately prior to pipetting verification for the instrument, therefore room temp and humidity should be the same.
Is room temperature and humidity a consideration in this method?
Each volume verification is tested a total of 3 times. If testing the volumes using the 96 pipetting head, a minimum of 3 96 well plates are dispensed. If testing the volumes using the Span 8 head, a minimum of 3 lanes of 8 well are tested. If testing a multi-dispense (aspirate once and dispense several times) then all of the dispenses for a minimum of three aspirations are tested.
Number of replicates
Pipetting verification method simulates the actual pipetting as closely as possible. For example, if liquid is dispensed into a dry well for the real method, the Orange G is also dispensed into the well first followed by water. If the liquid is dispensed into a well already containing liquid, the water is added first followed by Orange G.
Hints / Logistics not otherwise noted n/a
What Can You Do?
Request access to the NBS Molecular Resources Website from APHL
Fill out Molecular Assay Descript ion forms to describe your molecular assays
Describe your liquid handling solut ions for DNA Extract ion, PCR Set up or the On-Card Assay Method
NBS Molecular Resources Website
To add program members: Guisou [email protected]
Scientific questions and suggestions: Laura Hancock [email protected]
Molecular Assessment Program: Christopher Greene [email protected]
Molecular Subcommittee Questions: Michele Caggana [email protected]
For more information please contact Centers for Disease Control and Prevention
1600 Clifton Road NE, Atlanta, GA 30333Telephone, 1-800-CDC-INFO (232-4636)/TTY: 1-888-232-6348E-mail: [email protected] Web: www.cdc.gov
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
National Center for Environmental HealthU.S. Centers for Disease Control and Prevention
NBS Molecular Subcommittee:Michele Caggana (NY) – ChairpersonRichard Olney(CA)Stan Berberich (IA)Mark McCann (MN)Rachel Lee (TX)Sherly Pardo-Reoyo (PR)Mei Baker (WI)Anne Comeau (MA)
APHL:Guisou ZarbalianLaura RussellJelili OjoduMatthias Mart inMelissa Von Hatten
CDC:Suzanne CordovadoChristopher GreeneLaura HancockCarla Cuthbert
Use of trade names and commercial sources is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, or the U.S. Centers for Disease Control and Prevention.
, or the U.S. Department of Health and Human Services.
