KS USDA ABI7900 TaqMan Protocol, St. Amand, Revised Mar 26, 2010. Page 1 of 3

Basic Procedures for TaqMan probe assays on Toto, the ABI 7900HT RT System.

Notes:

•You MUST include non-template controls (NTC, samples with all PCR components minus DNA) as one or more of the samples, or you will be unable to analyze the results.

•You MUST use optical 384 well plates (bioexpress.com, T-3157-1).

•You MUST not write on the top of the plate.

•You MUST use optical-clear sealing tape, not mats, to cover the plates.

•Do NOT use any USB ports, disk drives, or USB disks to move files to Toto. Use the network available folder "Toto" to move data files to and from Toto. There is no anti-virus software on the Toto computer.

1. Master Mix. Set up the master mix as follows. The block has 384 wells and is not changeable. Total PCR volume must be 5 or 6 ul or the fluorescence readings will be incorrect.


2. AD Document. Create an Allelic Discrimination (AD) plate document from an existing example. See the Toto folder for examples. See the Allelic Discrimination getting started guide for more information.

a. Open the SDS software.

b. Select File > New Plate Wizard.

c. Select Allelic Discrimination (AD).

d. Create document from "Setup File". The setup file is your modified version of the sample file from Paul.

e. Review settings, sample names, and well assignments.

f. Save the file as an .sds file type.

3. Pre-read Plate. The plate MUST be read PRIOR to PCR to establish the amount of background fluorescence.

a. Turn on the 7900 and open the AD document created above.

b. Select Instrument > Plate Read > Connect.

c. Select "Open/Close" to open the instrument door.

d. Place the plate in the instrument in the proper orientation.

e. Click "Pre Read", reading only takes 3 minutes.

f. Select "Open/Close" to open the instrument door, remove plate if you will amplify the reaction on another PCR machine. Leave the plate in if you will amplify the reaction on the 7900.

4a. Option 1, for PCR on the 7900, AQ DOCUMENT and PCR. If you want to amplify the reaction on the 7900, then you will need to create a Standard Curve (AQ) plate document from an existing example. If you want to amplify the reaction on another PCR machine, then you do NOT need an AQ document. See the Toto folder for examples. See the Allelic Discrimination getting started guide for more information.

a. Open the SDS software.

b. Select File > New Plate Wizard.

c. Select Standard Curve (AQ).

d. Create document from "Setup File". The setup file is your modified version of the sample file from Paul.

e. Review settings, sample names, and well assignments.

f. Save the file as an .sds file type.

g. Select Instrument > Thermal Cycler, enter PCR volume (5 ul) & select standard mode.

h. Edit thermal profile to: Stage 1, 95°C for 5 min; Stage 2, 96°C for 15 sec, 60°C for 1 min, 60 cycles (40 does not work well for low quantity, low quality DNA).

i. Select Real-Time > Start Run.

j. Select File > Save.


4b. Option 2, for PCR on other machines, PCR. If you want to amplify the reaction on another PCR machine, then you do NOT need an AQ document. See the Allelic Discrimination getting started guide for more information.

a. Edit thermal profile on selected PCR machine to: Step 1, 95°C for 5 min; Step 2, 96°C for 15 sec; Step 3, 60°C for 1 min; Step 4, GoTo step 2 59 more cycles (40 does not work well for low quantity, low quality DNA).

b. Run PCR.

5. Post-read Plate. The plate MUST be read in order to record the fluorescence of each well. The pre-read background fluorescence will be subtracted from each well.

a. Open the SDS software.

b. Select File > Open and select your AD file.

c. Turn on the 7900 and select Instrument > Plate Read and press the "Connect".

d. If your plate is not in the 7900, press "Open/Close" and insert the plate.

e. Select "Post Read".

f. Press "Open/Close" and remove the plate.

g. Turn OFF the 7900.

6. Analyze results. If your data have well separated homozygous and heterozygous groupings, then the 7900 can automatically score each sample. If the groups are not well separated, you must manually score the groups. See the Allelic Discrimination getting started guide for more information.

a. Select File > Open and select your AD file.

b. Select Analysis > Analyze. The data will be scored.

c. Select the Results tab.

d. If the data are automatically scored, proceed to the next step. If the data are not scored, use the mouse to select data points and from the "Call" drop-down box, manually assign the allele calls.

e. Select File > Export and save your data report as a tab-delimited file for use with Excel.

f. Right-click on the plot to save the plot as an image file.

g. NOTE: when you save the AD document, the analysis results are NOT SAVED in the AD document. You must export the report.