Preparing of Template and Brews
1-Find the template to be sequenced from the -20 °C upright freezer and thaw it out in a cold water bath.
Templates need to be kept cold because extended exposure to room temperature will cause a decrease in sequence quality.
2-Make the product plate be arranging eleven eight-well strips on a Cetus 96 -well tray/retainer in columns 2-12. Remove a brew strip from the -20 °C standing freezer and place it in column 1. Make sure that the two empty wells in the brew strip lie in cells G1 and H1. Place the 96-well plate in a cold water bath to defrost.
Brews will quickly degrade at temperatures as low as 4 °C. Therefore, it is imperative that they be exposed to room temp as little as possible.
3-Centrifuge both the product plate and the template plate for 1 min at 1000 x g. ( Jouan Protocol #15 )
Air bubbles will cause Catalyst run failures. Centrifugation of the plates will cause the air bubbles to rise to the surface where they will not inter fere with the PCR.
4-Label the product plate with the corresponding template number followed by the number of times this particular template has been sequenced.
Prepping the ABI Catalyst 800
1-In the center of the machine is a series of Eppendorf tubes. Make sure that the tube in slot number one is filled with glycogen and that tube number five is filled with dH2O.
Make sure that the water tube is changed every time , and that the glyco gen tube is clean, as it has been known to grow molds. The glycogen will be used to precipitate the DNA at the end of the run.
2-Fill the brown bottle in the back-right corner with dH2O.
3-Check to make sure that the 95% EtOH bottle and the dH2O bottles to the left side of each machine are at least one-third full.
4-Check to make sure that the coolant bottle is at least one-quarter full.
5-Take the template and the product plates and place them in the left and cold right storage banks respectively. Make sure the covers are securely closed over on both banks.
Especially check the right cold storage lid on Kumquat to be sure that it is squarely on. Otherwise, the machine will ram the probe into the lid and destroy it.
Running the Machine
1-Double click on the Execdoc 2.0.1 application on the computer. Wait a few seconds and then click the Start button when it appears in one of the windows.
Clicking the start button will start the software transfer from the Mac to the Catalyst. This often takes a couple of minutes. However, this step is also prone to system failures. An easy way to tell if the computer isn't working is if a spinning wheel does not appear , does not spin or spins for longer than about four minutes. Refer to troubleshooting section for more details.
2-Two Catalyst windows will appear, a temperature and a log window. When directed by the log window, double click on the Execdoc Protocol Loader application - this application exists as an "EDP" icon on the desktop.
3-A new window will appear containing a menu of protocols for catalyst runs. Find the protocol you wish to use and double-click on it.
4-Execdoc Protocol Loader will then transfer the protocol you select into the Catalyst and subsequently another window will appear asking you if you want to start the downloaded protocol.
5-Click on the Start button to commence the Catalyst run.
Post-Catalyst Modification
1-Save the log file of the run into the appropriate log file. Use the following format: Month ( abb.) date, year ( abb.) <plate number>-<run num ber>; protocol ( no need to enter protocol if it is the full 96-well run ) .
The log files are a convenient form of record-keeping. If an error occurred during the please note it in the log book. If the error was a temperature problem be sure to save the temperature log as well.
2-Secure the aluminum tape to prevent desiccation and place the template in the -70 °C freezer in the appropriate drawer.
3-Spin the product plate at 3000 x g for 30 min ( Jouan Protocol #13 ).
4-Decant the alcohol layer and add 188 µ l of 70% EtOH into each well.
The Catalyst will add 95% alcohol to the product plate which will cause the DNA to precipitate out when centrifuged. The 70% EtOH wash is to clean up the DNA.
5-Centrifuge the sample at 3000 x g for 10 min ( Jouan Protocol #14 ).
6-Decant the EtOH by inverting the plate over the sink. Look to see that there is a white pellet at the bottom of each well.
7-Vacuum dry the sample either in the speedvac.
8-Place the sample in the "To be Sequenced " area of the refrigerator.