Step 1: Installing the CounterTrace software
- Create a new folder called CounterTrace on your computer.
- Copy the CounterTrace.exe and ctw.chm files to the CounterTrace folder.
Step 2: CounterTrace Spectral Calibration
- Add 3 µl of 1000x CounterTrace Loading Additive to 997 µl of sterile water (3x final conc.) or 0.1 mM EDTA pH7.4
- Aliquot 10 µl into each well of a 96-well plate, or one quadrant of a 384-well plate. For 48 capillary array ABI 3730 DNA sequencer use only 48 position wells.
- Run the plate on an ABI 3730 sequencer using either one of the following modules and run times:
- StdSeq36_POP7 module with a run time of 2800 seconds (36cm capillary arrays).
- LongSeq50_POP7 module with a run time of 6100 seconds (50cm capillary array).
- For the Applied Biosystems 3700 DNA sequencer with a 50 cm capillary array use either one of the following modules and run times:
- POP5DEFAULT module with a run time of 3 hours
- POP6DEFAULT module with a run time of 4 hours
- Once the plate run has completed, choose “CounterTrace Spectral Calibration” from the “Process” menu of the CounterTrace software and select the folder containing the calibration (standard only) traces and press “OK”. A CounterTrace spectral calibration file called machines.ctc will be created after approximately 30 seconds in the CounterTrace folder.
Step 3: Determining the optimal CounterTrace Loading Additive concentration:
- Perform 30 standard DNA sequencing reactions using a control DNA template (e.g. pGEM-3Z). Clean the reactions using your normal sequencing clean-up protocol.
- For each 10µl of cleaned DNA sequencing reaction add 2 µl of CounterTrace Loading Additive (CLA) diluted to one of the following concentrations: 6x (1 µl CLA + 167 µl H20), 12x (2 µl CLA + 16 6µl H20), 18x (3 µl CLA + 165 µl H20), 24x (4 µl CLA + 164 µl H20) and 30x (5 µl CLA + 163 µl H20). With each CLA concentration make six reaction replicates (i.e. six of the 6x, 12x, 18x, 24x and 30x dilutions).
- Run the DNA sequencing reactions using the sequencer run module and times used in Step 2.
- Once the run has finished, choose “Add Trace Folder” from the “File” menu and select the run folder containing the 30 trace files. Choose the output folder from the “File” menu. Click “Start” in the “Process” menu to process the trace files.
- After the traces are all processed, look at the status panel and identify the lowest CounterTrace Loading Additive concentration at which the fewest “Can’t find standard” errors occur. This concentration is the optimal CounterTrace Loading Additive concentration for your samples. For example, if 18x, 24x and 30x all gave the lowest number of errors then the 18x concentration is optimal.
Step 4: Running CounterTrace on your samples
- Add the optimal CounterTrace Loading Additive concentration as determined in Step 3 to your cleaned production DNA sequencing samples and run on the sequencer using the run module and time used in Step 2.
- Process the trace files with the CounterTrace software as described in Step 3.4. Compare the sequencing files before (run folder) and after (output folder) processing to determine the improvement in read length and sequence quality provide by CounterTrace.