Scripts and reagent cassette modifications needed to obtain longer reads on the 454/Roche GS-FLX without the XLR reagent update
Doug White and Graham Wiley
Roe Lab, University of Oklahoma
September 20, 2008
Installing the 126x FLX run scripts
1. The 126x run script is available here
2. Upload the expanded .icl run scripts on to the FLX computer into the directory:
that contains the default 42x and 100x .icl run scripts.
3. These new .icl files do not show up in the run program:
4. To run them, start the sequencing run as usual and select the default 100 cycle run script, 100x_TACG_70x75_LR70KIT.icl.
5. Continue as usual until you get to the "Insert new PTP" screen. At the bottom of the window, the blue, active link "Script" followed by the script name, should be highlighted.
6. Click on "Script" and a small window will open showing the contents of the runScripts directory.
a. Select "70x75 and then "TACG" to see all of the .icl files
b. Select the 126x_TACG_DDW.icl run script and click OK.
c. Start the sequencing run.
7. By the way, this is the same way to select the "service" directory to run a volume verification or one of the pump calibration scripts.
Reagent cassette modifications, brief protocol:
1. Before adding the DTT to the four 1L buffer bottles, add 2.5ml of Buffer CB to each of the Substrate, Inhibitor, and Apyrase tubes.
2. Add 174ul of apyrase (instead of 164ul) to the apyrase tube to maintain the original concentration.
3. Transfer 100ml from the buffer3 bottle to the buffer0 bottle.
Reagent cassette modifications, detailed protocol:
Before beginning the protocols described below, complete the preparation of the picotiter and the pre-wash run as described by Roche. Then, follow the buffer thawing and cassette loading below, noting that only steps 7, 8, 11, 18 and 19 indicated in bold italic, differ from those described in the Roche manual