DNA Quantitation and Normalization Procedure
A. DNA Quantitation by Hoechst Dye
Disclaimers:
Hoechst dye is potentially carcinogenic – wear gloves and dispose of waste properly.
Hoechst dye is light sensitive, keep from light and store frozen.
Materials Required:
- Hoechst dye (bis-benzimide, Sigma B2883) solution, 1 mg/ml in water. Keep frozen between uses.
- DNA standards. A separate plate must be prepared with standard DNAs of known concentrations.
The standards must be in the leftmost columns (e.g., columns 1-4). To get the most accurate readings,
use standards close to the expected concentration of your DNA samples and use 4 replicates for each
concentration. The assay can be used for DNA concentrations ranging from 5-200 ng/µl. It is convenient
to have a plate of standards at varying concentrations prealiquoted into Axygen PCR plates in columns 14. The default format for the standards for the normalization program is: A1-A4: 0 ng/µl, B1-B4:10 ng/µl,
C1-C4: 25 ng/µl, D1-D4:50 ng/µl, E1-E4: 75 ng/µl, F1-F4: 100 ng/µl, G1-G4: 150 ng/µl, H1-H4:200 ng/µl.
- 10X TNE buffer -100mM Tris pH 7.5, 1 M NaCl, 10 mM EDTA
- Black 96-well microplates: Greiner Black Polystyrene, flat bottom
Instructions for the Hoechst Assay:
1) Thaw Reagents, mix well.
2) Dilute Hoechst Dye to 2µg/ml in 1X TNE Buffer. You will need ~10 ml/plate plus ~30 ml extra for the
reservoir. Mix well. Keep diluted dye from light by covering with foil.
3) Add Diluted Hoechst 95ul/well to black assay plates using the Biomek FX. Use the appropriate
quant1 program from the FX's Methods\DNA Quant folder based on the number of plates you wish to fill.
Make one assay plate for each sample plate plus one extra plate for the standards, e.g., if you have 2
sample plates make 3 assay plates using the quant1_3 plate program. Use 250 ul unfiltered tips and the
inverted tip lid as the reservoir.
4) Add 5 ul/well of standard or unknown DNA using the proper FX's quant2 program to suit your plate
type. Normally the standards are in Axygen PCR plates. This program uses the 250 ul unfiltered tips.
5) Read the plates immediately after adding the DNAs.
Set the Tecan SpectraFluor Plus (Xfluor4) with these settings before reading:
Click on Xfluor4...Edit Measurement Parameters.
General: Fluorescence
Plate: GRE96fb.pdf
Measurement Parameters: Other; Excitation 360 nm, Emission 465 nm; Read from TOP
Manual Gain = 95
Integration Time: 40 usec; Flashes = 3
Click OK to close Measurement Parameters
Click on Xfluor4…Default Result Presentation. In the Destination box select the “Attach at the end of the
active worksheet option”.
Click OK to close Default Result Presentation.
Click on Xfluor4...Start Measurement.
(Note: if the proper Excitation or Emission values are not in the drop-down menus you will need to change
the filter sets in the machine. Choose Xfluor4…Move plate and filter… and then choose the appropriate
filter to Move Out. The filters are located in the drawer under the machine).
6) Read the Standards plate first. Then read your other plates after that without changing settings.
7). Next, you must add your plate names to the Excel file. You will see that above the output for each
plate is a cell that says “Rawdata”. Change this cell to read “Rawdata:platename” using the platename
of your choice. For example, if the name of your plate is ABC001, change this cell to read
Rawdata:ABC001. Make sure you label each of your sample plates properly as this information will be
used to generate the normalization files. Label the standards as “Rawdata:standards”.
8). Save the file as a tab delimited file on the HIPfileserver. Choose File…Save As… then at the bottom
click on the “Save as Type” and choose the “Text (Tab delimited) (*.txt)” option. Click Save to save.
B. Using this Normalization Program:
http://128.103.32.197:8080/DNANORM/
1). Enter the proper parameters for the normalization computation.
Standards
Number of columns: Enter the number of columns of standards that you used. The default value is 4
columns of standards (columns 1-4 on the plate).
Rows from: Enter the location of the rows that you wish to use for calculating the standard curve.
Choose rows corresponding to DNA concentrations close to the concentrations of your samples. You can
get a rough idea of which values of the standard curve to include in the calculations by comparing the
relative fluorescence values for your samples versus the values obtained with the standards. The default
value is 1 to 6 corresponding to rows A through F, since typical DNA miniprep concentrations are up to
100ng/ul if the typical standards are used. If you have DNA concentrations that are higher you might wish
to choose rows 4 to 8.
To aid you in visualizing the standard curve, a simple Excel spreadsheet for plotting the standard
curve can be downloaded from this page. This spreadsheet can also be found in \DNAnorm directory on
the HIPfileserver as “Standard Curve Plotter.xls”. (Please do not save your values in this file in the
DNAnorm directory).
Standard DNA concentrations in ng/ul: Enter the DNA concentrations (ng/ul) that you used in your
standards for the samples that you want included in the assay. The concentrations should be separated
by a comma. The total number of concentrations listed must correspond with the total number of rows
chosen in ‘Rows from’ above. For example, if you have chosen rows from 4 to 8 above you must have 5
sample concentrations listed in this section.
Options
Estimated source DNA volume in ul: Enter the volume in microliters of DNA that you have in your
sample. It does not need to be exact.
Normalized DNA volume in ul: Enter the amount, in microliters, of the final volume of DNA that you
wish to have after normalization.
Normalized DNA concentration in ng/ul: Enter the desired concentration of the DNA after
normalization (ng/ul).
Source DNA container type: Choose the plate that your source DNA is in.
Costar 96 well flat bottom TC plate = Costar96flatb on 30mmNest/MP16
Costar 96 round bottom (old glycerol stock plate) = Costar96ub on 30mmNest/MP16
Greiner #650101 u bottom (glycerol stock plate) = Greiner96ub on 30mmNest/MP16
Axygen PCR plate = PCR96 on 30mmNest/MP16
RKRiplate Deep Well block = RK96dw/MP16
Abgene 800 ul conical bottom = Abgn08mlDWonMP16
Greiner 300ul conical bottom =Greiner96vb on 30mmNest/MP16
Normalized DNA container type: Choose the type of plate that you will use to contain your normalized
DNA (destination plate).
Water Container type: Currently the only allowable reservoir is the single chamber deep well reservoir:
Reservoir/MP16
Barcode label for water reservoir: The plate containing water needs to have a barcode on it for the
Tecan to recognize it. The default is having the barcode as “Water”. (Don’t forget to capitalize the W).
Upload the Hoescht Dye Assay file: Use the browse function to locate the file generated by the plate
reader. Typically, this will be saved to the HIPfileserver drive. As noted above, the file must have the
plates properly labeled and be exported as a tab-delimited file.
Provide a string to identify your output files: Choose a label for the files that will be created by this
program. Typically, this might be your name and the date. Do not have spaces or special characters in
the string (hyphens and underscores are OK). Example: yourname_Jan_1_2008.
2).
Output. Click Submit and the program will return a page that lists the parameters of the
normalization. These include the slope and intercept of the standard curve, as well as the correlation
coefficient, R. Generally, the R value for the standard curve should be between 0.99 and 1.0.
The program generates the predicted values of each of the standards using the standard curve,
as well as the percentage error in the calculations between the predicted and observed values for these
standards. This information is useful to determine the range of sample DNA concentrations for which
your standard curve will be accurate. Generally, you will want your sample DNA concentrations to be
within the range of the standards that is predicted to an accuracy of ±10%. If your standard values are
greater than this range you may want to choose incorporate different standards in the calculations and
repeat the submission process.
The program will also create 6 types of files on the HIPfileserver (Hipfs:), their names are listed at
the bottom of the output page. The files will be located in the \DNAnorm directory. Note: Please use this
directory only as a temporary place to store your files. Files left in this directory after one week may be
erased! In the future these files will be emailed to you instead of put in the DNAnorm directory.
If your experiment requires more than 5 plates of DNAs to be normalized: The Tecan robot can
hold only 5 sets of DNAs for normalization at a time (5 source plates, 5 destination plates, plus water
reservoir). If your Hoechst data contains more than 5 sample plates the program will create multiple
normalization files with the first 5 files in the first normalization, next 5 in the second normalization, etc.
Using the sample string from above, the files generated by the normalization are:
yourname_Jan_1_2008_src.xls This file contains the DNA concentrations on your source plate.
yourname_Jan_1_2008_dest.xls This file contains the DNA concentrations on your destination plate
after normalization.
yourname_Jan_1_2008_output.xls This file contains the linear tabulation of the amounts of DNA and
water added to each well for the normalization.
yourname_Jan_1_2008_normalization.log This is a text file that contains information about the
normalization process generated by the program. The file contains the names of the plates that you will
need to have on the robot deck during the normalization. It also contains information on the standard
curve used to calculate DNA concentrations.
yourname_Jan_1_2008_barcode.txt This is a text file that contains the names of all of the plates that
need barcoding during the normalization. You can import this file to the barcode printer to print out your
barcodes.
yourname_Jan_1_2008_worklist_1.gwl This is the actual worklist file that must be uploaded to the
Tecan robot. Since only 5 plates of DNA can be normalized at one time a worklist is generated for every
5 plates. For example, if you input 10 plates for normalization you will have two .gwl files created. They
will be listed with the suffixes _worklist_1.gwl and _worklist_2.gwl.
C. Barcoding. You must barcode your plates properly for the Tecan to identify them. The
normalization program generates the worklists using the plate names that your entered in the Excel file
(whatever you entered after “Rawdata:”) as source plate names and adds the suffix “-norm” for the
normalized plate names. For example, if you had a source plate named “plate1” the program will expect
the destination plate to be barcoded as “plate1-norm”. Note: The plates must be barcoded exactly as
listed in this file or the robot will not use them. This includes matching the capitalizations and spaces.
The _barcode.txt file generated during the normalization contains all of the plate names needed
for the normalization process. This is a text file and can be used to print out the barcode labels at the
Zebra barcode printer in the robot room.
Affix the barcodes to your plates on the right side (i.e., adjacent to column 12). The barcoded
area (not the text) of the label should be centered in middle of the plate in the left-right dimension and the
top of the label should be aligned with the top edge of the plate. Plates with improper positioning of the
label may not scan properly during normalization.
D. Using the Tecan Robot to do the normalization.
If you have not done this procedure before it is suggested you check with Mauricio to get trained on use
of the Tecan before starting. The following instructions are for a ‘cold start’, if the machine has been in
recent use you can omit the first two steps and go directly to step #3.
1). Make sure the robot is turned on (rocker switch at top right)
2). Start the robot control software by clicking on the Gemini icon on the desktop.
The Gemini software requires a login, this should work automatically. If it doesn’t, use the logon
info which Mike has attached to the LCD computer display. [The login info for the computer (the Windows
logon) is also pasted onto the LCD computer display in case you need to restart the computer.]
3). Close the rearray program that loads with the Gemini software by clicking File…Close. Open the
normalization program by clicking Open… and choosing C:\Gemini\In Development\Plates on MP16 and
30mM Nests\Plates on MP16.
4). The instrument should be flushed and reinitialized before use. Make sure the 4 liter flask under the
robot is filled with tap distilled water. If you are doing a ‘cold start’ make sure the robot deck is clear as
the robot arms will move during reinitialization.
Start the flush by clicking on the “flush instrument” icon. This is the 11 th icon from the left, it looks like a
green pipette tip. (If you hover the cursor over the icon it will say “flush instrument”.)
This will bring up a dialog box, click OK to accept the default values and start the flushing. The machine
will say “Initializing Instrument” and the arms will move.
Repeat the flush until no bubbles are observed in the tubing leading to the pipetter arm.
5). Load the Worklist. Double-click on the second line of the program which is titled, “Worklist”. This will
bring up a dialog box.
Choose “Load Worklist”.
Worklist (Path and Name): browse to the HIPfileserver to load your worklist from the \DNAnorm
or other directory.
Leave the boxes all checked to use all of the pipetters.
Liquid Class: Choose either “DNA Mix” or “DNA No_Mix” as our your liquid class depending on
whether you want your source DNAs mixed prior to the procedure.
Click OK to close the dialog box.
6). Next you need to set the starting position for the pipette tips. Choose Execute…Set DITI position.
Choose DiTI200, 96 tips loading rack.
Chose Next DITI position: “200ul-1”.
Position in rack: 1.
These settings tell the robot you are using 200ul size tips and starting with a new, full box of tips. (The
machine numbers the tips 1-96 with A1 being position 1, B1 is position 1, A2 is position 9, etc). Click OK.
7). Click on the blue checkmark icon to validate the program. If successful it should say “No errors
found”.
8). Open the output file and delete any previous entries. This file is a log file that contains the names of
plates scanned in by the robot, you want to delete out previous entries so you will see only the files
scanned in during your run. Do this by double-clicking the “Shortcut to Output” icon on the desktop
(Alternatively, you can go to “My Computer.. then choose C:\Gemini\Output to open). If the previous user
has left any plate names in this file select and delete them. Minimize this file (do not close) by clicking
the minimize icon on the top right so you can return to it later easily.
9). Load the tips. The tips used for DNA normalization are BioRobotix MBP 200ul non-sterile tips,
catalog #903-262. The rack of tips has a notch on one side of the purple plastic, orient the rack so the
notch is at the top Left side. The rack has a plastic rim that goes on the outside of the horizontal metal
bars on the robot. The rack needs to click into the holder, do this first tucking the right side under the clip
then push the left side of the rack down until it clicks in place.
Make sure the tip shoot is clear and the bin underneath it is not overflowing
10). Set up the robot deck. Most source and destination plates must sit on the white delrin “nests” to
make them sit higher on the deck, some plates must sit on the polypropylene 96 well plates left near the
robot. The type of plate must be matched to the proper nest according to the table below:
Require White
Delrin Nests
Axygen PCR plates
Greiner 300 ul
Conical plates
Costar 96 well
Flat bottom
Costar 96 well
Round bottom
Greiner 96 well
Round bottom
(glycerol stock plates)
Any plate less than
300ul in size
Require Matrix 96well plate Nests
Abgene 800 ul
Do not use Nests
Other 800 ul plates
Other 2ml size plates
Water reservoir
RK Riplate
(Deep well blocks)
The plates can be put in any order on the deck but must be in the correct orientation with cell A1
at the top left. The barcodes must be on the side facing right. If the deck is set up properly the tops of all
the plates should be at a similar height.
11).
Start the procedure. Click the green arrowhead icon at the top of the program to start the robot.
This will bring up a dialog box that says “How many plates are on the deck?”. Enter the correct number of
plates, including all of the source plates, destination plates, and the water reservoir, then press OK. The
robot will begin to scan the barcodes on the plates. You may need to lift the right side of the plates up or
down slightly to get the scanner to read the plate. Once the scanner has read the plate it will move to the
next plate.
A message box will come up stating “Check which plates have been read by the scanner”.
Determine which of your plates have been scanned properly by clicking on the output file at the bottom
that you minimized previously. If all of your plates are listed properly, delete them from the file, close the
file and click OK on the message box and the robot will start the process.
If one or more of your plate names are missing you must start over and rescan all of the plates.
First, click Cancel in the message box. Then, delete all of the plate names from the output file and
minimize the file again. Finally, restart the procedure by clicking the start button again. For your run to
work you must have all of the plate names scanned in properly. The scanner can be flaky if it gets out of
alignment so may need multiple tries to get it to work properly, ask Mauricio for help if your plates are not
scanning properly.
12).
The program will run to completion in about 15 minutes per plate. The correct amounts of water
will be added to each well first then the correct amounts of each DNA will be transferred. You will be
prompted to change the tips if necessary.
When you are done seal your plates and remove them from the deck. The tips are expensive so
save any unused tips for later use by putting them back into their wrappers and sealing them up (you will
need to define the proper starting position for reuse under step 6 above).