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3730xl Analyzer Protocol

Posted by shinegene, 01 June 2009 · 1,514 views

molcecular biology
ABI 3730xl Setup & DNA Sample Loading

NOTE: See Spring 2005 Instrument Configurations for sample plate loading matrix

3.1 Start the Instrument:

NOTE: In most cases, the instrument will be running. Sample loading will begin at step d below.

a. On the instrument, ensure that the:

i. Oven door is closed.

ii. Instrument door is closed.

iii. Stacker Drawer is closed.

iv. Buffer, water, and waste trays are loaded.

b. Make sure that the computer is on and logged in as the proper ABI instrument.

c. If the instrument is not on, press the on/off power button located on the front of the instrument. Ensure that the green instrument status light is on and constant before proceeding (this takes about 1 minute).

i. The light on the left hand side is the instrument status light. It is solid green when the instrument is “ready,” it blinks orange when the instrument is initializing, and it blinks red when there has been an instrument failure.

ii. The light on the right hand side is the stacker light. It is solid green when the stacker door is closed and blinks green when the stacker door is open.

d. Prepare the Instrument and Loading DNA Sample Tray Assemblies (standard 3kb and 8 kb production plates)

NOTE 1: 3kb and 8 kb sample plates will be stored in bins in the ABI Backlog labeled “1/32 BigDye,” “1/16th BigDye,” or “1/10th BigDye”

NOTE 2: See Spring 2005 Instrument Configurations for a list of standard 1/32nd 1/16th and 1/10th dedicated instruments. All instruments run the standard 15S protocol, but the varying BigDye concentration plates are assigned to specific instruments to monitor the affect of the different chemistries on array performance.

i. Make sure that the instrument’s green status light is on (either flashing or constant).

ii. Make sure that adequate levels of buffer and water are in the appropriate reservoirs.

iii. Check the level of POP-7 polymer in the bottle to ensure sufficient volume for run.

iv. Scan in the generic ABI barcode on the side of the DNA sample plate into the Sample Sheet Manager.

Important! Make sure that it is successfully imported into the Sample Sheet Manager and that the proper number of quadrants is selected at the scan step.

v. Pull open the stacker drawer. The stacker light flashes green.

vi. Open the metal door of the In Stacker tower and place up to 16 (maximum) tray assemblies into the stacker (refer to the loading guide posted in room 150 or see Appendix A).

Important! Ensure that the tray assembly is fully assembled (both retainer clips are fully fastened to the base) and that it sits flat in the In Stacker. Failure to do so may result in instrument crash.

NOTE: The bottom tray will run first.

vii. Close the metal In Stacker door and the stacker drawer. The stacker light becomes a constant green.

NOTE: If the instrument is not already running a plate, then once the plates are loaded into the In Stacker and the Stacker drawer is closed, “Unknown” appears in the Data Collection Viewer’s Input Stack window and the “Play” button on the top toolbar turns green.

viii.If the instrument is currently running when you add plates to the In Stacker, there is nothing left to do. The instrument will automatically run plates until the In Stacker is empty. If the instrument is not processing plates, click on the green RUN button.

ix. Click on the OK button to start processing plates.

e. Prepare the Instrument and Loading DNA Sample Tray Assemblies (Fosmid plates)

NOTE 1: Fosmid plates will be stored in the ABI Backlog in a specially marked “Fosmid” bin. The JGI barcode will be marked with a vertical green stripe.

NOTE 2: certain instruments are near-dedicated instruments for processing Fosmid plates. These instruments have a modified run conditioned installed as their default for processing Fosmid plates. Fosmid plates should be loaded exclusively on these instruments unless additional sequencing capacity is required. See Spring 2005 Instrument Configurations for a list of fosmid dedicated instruments.

NOTE 3: Fosmid plates should never be loaded on an instrument that has 3 or more sample plates already loaded unless the Fosmid plates are loaded to the bottom of the in stack.

i. Follow steps i-ix of d. Prepare the Instrument and Loading DNA Sample Tray Assemblies above.

f. Prepare the Instrument and Loading DNA Sample Tray Assemblies (standard 3kb and 8kb production plates on Fosmid-dedicated instruments)

NOTE: Standard production plates should only be loaded on fosmid-dedicated instruments if there are not enough Fosmid plates available to keep these instrument running continuously.

i. Follow steps i-iv of d. Prepare the Instrument and Loading DNA Sample Tray Assemblies above.

ii. After scanning in the sample plate, select the Plate Manager link from the left hand frame of the Unified Data Collection software screen.

iii. Scan the generic barcode into the Scan or Type plate ID field.

iv. The plate name will appear in the field below. Select the plate and hit the Edit key on the bottom of the screen. The Sequencing Analysis Plate Editor window will appear.

v. Select the cell found at row A1 under the column heading Instrument Protocol. A drop down menu will appear. Select the 15S instrument protocol.

vi. Click on the column heading titled Instrument Protocol to select the entire column. Hit Ctrl+D on the keyboard. This will change the Instrument Protocol for the entire row to 15S.

vii. Follow steps v-ix of d. Prepare the Instrument and Loading DNA Sample Tray Assemblies above.

g. Prepare the Instrument and Loading DNA Sample Tray Assemblies (Fosmid plates on standard 1/16th or 1/32nd instruments)

NOTE 1: Fosmid plates should only be loaded on 1/32nd or 1/16th instruments if there are too many Fosmids to process on the near dedicated instruments.

NOTE 2: Fosmid plates should never be loaded on an instrument that has 3 or more sample plates already loaded unless the Fosmid plates are loaded to the bottom of the in stack.

i. Follow steps i-iv of f. Prepare the Instrument and Loading DNA Sample Tray Assemblies (standard production plates) above.

ii. Select the cell found at row A1 under the column heading Instrument Protocol. A drop down menu will appear. Select the FOS instrument protocol.

iii. Click on the column heading titled Instrument Protocol to select the entire column. Hit Ctrl+D on the keyboard. This will change the Instrument Protocol for the entire row to FOS.

iv. Follow steps v-ix of d. Prepare the Instrument and Loading DNA Sample Tray Assemblies above.

h. Ensure that the Out Stack is routinely emptied of processed trays (once per day) so that it will not overflow and stop the instrument.

i. Once the plates have been removed from the Out Stack, go to the Venonat database, click on ABI3730 DB à Scan to Trash (Using ABI Barcodes) form à scan in the plates’ generic barcodes to verify that all plates have been fully processed.

NOTE: If a plate has not been processed, seal it with a clear seal and place it in the bin labeled “Plates not posting” in the deli refrigerator in room 147. Plates may take up to one hour to post.




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