ABI PRISM® 310 Genetic Analyzer Test CCD 4
Contents
1. Test CCD 4 Color Run as a Stand alone Module NOTE The Test CCD 4 Color Run must be performed on a properly installed and polymer filled capillary If a capillary is not present see the Capillary Install section of this document If the capillary is not filled with polymer see the Performing the Seq Fill Capillary Run section of this document Step Action o O 1 __ Ensure that the instrument is on and that a filled capillary is properly installed 2 If Data Collection software is not on manually launch it 0 ie els el ala configurations appropriate for your sample and plate configuration In the new sample sheet window enter Test CCD 4 Color for the name of the sample If you are creating a Sequencing sample sheet Sequence Analysis Sample Sheet e Select a dye set primer file P Semple Name Dyeset Primer oa fi eee e Selecta matrix file pel RAS e Save the sample sheet as Test CCD 4 Color If you are creating a Genescan Software sample sheet e Save the sample sheet as Test CCD 4 Color Close the sample sheet window when finished Page 1 of 7 Test CCD 4 Color and Seq Fill Capillary Module Applied Biosystems From the File menu select New and then click on either Sequence Injection List or Genescan Software Injection List according to the sample sheet made in step 4 6 From the Sample Sheet drop down list select the sample sheet made in step 4 This will fill in the injection2. property of their respective owners Page 7 of 7 Test CCD 4 Color and Seq Fill Capillary Module
3. Applied Biosystems To navigate within this document use the 4a gp Or g keys ABI PRISM 310 Genetic Analyzer Test CCD 4 Color and Seq Fill Capillary Run Modules BEFORE PERFORMING ANY TROUBLESHOOTING WORK ON YOUR ABI PRISM 310 GENETIC ANALYZER PLEASE READ THE INSTRUMENT USER S MANUAL FOR SAFETY AND WARRANTY INFORMATION AND FURTHER DETAILS ON USE OF THE SYSTEM NOTE Text in this fashion indicates a link to a picture or another section of this another document Please contact AB Technical Support if you have any questions regarding this procedure About the Test CCD 4 Color and Seq Fill Capillary Run Modules The ABI PRISM 310 Genetic Analyzer collects fluorescent signals from dye labeled fragments passing through a polymer filled glass capillary Data quality can be affected if the capillary window is dirty or improperly positioned The Test CCD 4 Color run detects the cleanliness and quality of the capillary The Seq Fill Capillary run flushes fresh polymer through the capillary When to Perform the Test CCD 4 Color and or Seq Fill Capillary Run Modules Test CCD 4 Color e Every time a new capillary is installed on the instrument e Anytime a used capillary is reinstalled repositioned on the instrument Seq Fill Capillary To prime a new capillary If the Test CCD 4 Color Run results are not optimal If noisy elevated baseline is encountered during a run To check the system for polymer leaks Performing the
4. d Biosystems Performing the Seq Fill Capillary Run NOTE The Seq Fill Capillary run must be performed on a properly installed capillary If a capillary is not present see the Capillary Install section of this document Make sure to have a 1 5 mL Eppendorf tube with the cap cut off present at position 3 on the autosampler Fill the tube with sterile deionized water Step Action 1 Ensure that the instrument is on and that a capillary is properly installed 2 lf Data Collection software is not on manually launch it 3 From the File menu select New and then click on one of the four sample sheet configurations appropriate for your sample and plate configuration In the new sample sheet window enter Seq Fill Capillary for the name of the sample If you are creating a Sequencing sample sheet uae A AE TETEE e Select a dye set primer file he lear e Selecta matrix file EE a e Save the sample sheet as Seq Fill Capillary If you are creating a Genescan Software sample sheet e Save the sample sheet as Seq Fill Capillary Close the sample sheet window when finished From the File menu select New and then click on either Sequence Injection List or Genescan Software Injection List according to the sample sheet made in step 4 From the Sample Sheet drop down list select the sample sheet made in step 4 This will fill in the injection list with the name of the sample From the Module drop down list for this sampl
5. e select Seq Fill Capillary The autosampler will move the waste vial position 3 to the capillary The syringe pump will then proceed to pump polymer through the capillary Approximately 4 7uL of polymer will be flushed through Page 6 of 7 Test CCD 4 Color and Seq Fill Capillary Module Applied Biosystems Contacting AB Technical Support By Telephone 1 800 831 6844 By Internet http Awww appliedbiosystems com support Then click on Frequently Asked Questions and then the Ask a Question tab By E mail AB Technicalsupport appliedbiosystems com Manually Launching Data Collection Software Double click on the shortcuts on the computer desktop Alternatively navigate via the Apple a r Menu Start Menu A a T A T E EEC ABI Prem 310 Collection bat On the PC On the Macintosh computer For Research Use Only Not for use in diagnostic procedures Notice to Purchaser License Disclaimer Purchase of this software product alone does not imply any license under any process instrument or other apparatus system composition reagent or kit rights under patent claims owned or otherwise controlled by Applera Corporation either expressly or by estoppel Applied Biosystems ABI PRISM and GeneScan are registered trademarks and AB Design and Applera are trademarks of Applera Corporation or its subsidiaries in the US and or certain other countries All other trademarks are the sole
6. e lines should not be sloping wavy spiked or rounded Position All four baselines should be under 2000 rfu s Typical baselines will range from the low hundreds 400 600 to the low thousands 1100 1500 Typically blue and green are the lower baselines while yellow and red are the higher baselines This is not an absolute rule depending on the laser in use This Test CCD 4 Color result left shows four excessively high baselines 8000rfu It was generated as a result of improper positioning of the capillary in the capillary holder REJECT testsamplename 1 Al This Test CCD 4 Color result left shows relatively low baselines 200rfu The lines are compressed on one another and are difficult to distinguish REJECT This Test CCD 4 Color result left shows drastically sloped baselines The lines are actually exceeding the high range of the window This was generated by an dusty capillary window REJECT Page 3 of 7 Test CCD 4 Color and Seq Fill Capillary Module SX Applied D Biosystems a a A a This Test CCD 4 Color result left shows a slight sloping early on in the test This is a normal function of the laser optics There are four distinct lines They become relatively horizontal The signal range is from 500 1000 ACCEPT Troubleshooting Bad Test CCD 4 Color Results St
7. ection 2 Fill in the Tube amp Sample Name for Injection 1 by selecting any one of the o mme sample names e a a a ee A 45 sample PI E ES mo BEJ Inj Tube amp Sample Secs k kY C Timej niz P me CES a eee ee E A1 sample 1 Seq POPS 1 mL E P Arama sa 4S sample 3 Seq POPS 1 mL E gt sa 12 2 Fill in the Module for Injection 1 by selecting Test CCD 4 Color Fill in the remainder of the modules for the run if they have not been auto filled NOTE The Test CCD 4 Color module does not actually inject any sample Therefore do not be concerned about using any sample as a result of this procedure m Page 2 of 7 Test CCD 4 Color and Seq Fill Capillary Module Applied Biosystems Step Action 7 Click Run to start the Test CCD 4 Color with an existing sample sheet The laser will excite the detection cell window and gather data As the run progresses examine the scan line information Compare your results with the criteria in the evaluation section below 8 After the Test CCD 4 Color is finished the rest of the run will proceed Evaluating the Test CCD 4 Color Results To evaluate the results of a Test CCD 4 Color run apply the following THREE rules Number The Test CCD 4 Color run should generate FOUR distinct baselines The four baselines are Red Blue Black and Green Flatness The four baselines should be relatively FLAT meaning the lines are horizontal Th
8. ep __ Action j 1 Carefully clean the capillary window with a lint free lab wipe dampened with clean 70 ethanol and re position the capillary back into the detector window Repeat the Test CCD 4 Color 2 Perform the Seq Fill Capillary run then repeat the Test CCD 4 Color 3 Install and prepare a new capillary and repeat the Test CCD 4 Color 4 Contact AB Technical Support if problems persist Capillary Install 1 Open the instrument doors Open the heat plate door and the detector window door Remove a capillary from the curved plastic tube Partially unscrew the capillary fitting p n 005404 e on the right side of the pump block and thread the i capillary through the fitting NOTE The end of the capillary closest to the colored dot clear window section goes to the pump block Kg b J Begin tightening the capillary fitting while keeping the capillary end of the capillary is to the right of the vertical AE channel in the block and well to the left of the tip of the capillary fitting NOTE Due to the compressive forces the end of the capillary in the block will move slightly to the left as the fitting tightens Make sure the end does not enter or cross the vertical junction NOTE 2 Leave the free end of the capillary loose while the detection side is being secured to the block Page 4 of 7 Test CCD 4 Color and Seq Fill Capillary Module Applied Biosystems Action Once the capi
9. list with the name of the sample From the Module drop down list for this sample select Test CCD 4 Color The completed injection list will look like the example above 8 Click Run to start the Test CCD 4 Color stand alone run Z Z o Z The laser will excite the detection cell window and gather data As the run progresses examine the scan line information Compare your results with the criteria in the evaluation section below Performing the Test CCD 4 Color Run with an Existing Sample Sheet NOTE The Test CCD 4 Color Run must be performed on a properly installed and polymer filled capillary If a capillary is not present see the Capillary Install section of this document If the capillary is not filled with polymer see the Performing the Seq Fill Capillary Run section of this document From the File menu select New and then click on either Sequence Injection List or Genescan Software Injection List according to the samples you will be running From the Sample Sheet drop down list select the sample sheet that you plan to run This will fill in the injection list with the sample names of the samples you plan to run NOTE This step presumes that you have already filled out a sample sheet Highlight row 1 of the injection list and Aes P then from the Edit menu select Insert This will create a blank row above row 1 What was previously Injection 1 will now E be Inj
10. llary is secure pull slightly on the capillary It should not move Carefully clean the capillary window with a lint free lab wipe dampened with clean 70 ethanol n NOTE Rub this area vigorously but minimize bending the capillary window This ensures any particulates dust or contaminants are removed window The colored dot above the capillary window should rest at the top border of the detector window plate With one hand keeping the capillary in place close the detector window door with the opposite hand Carefully position the capillary into the detector Affix a small piece of thermal tape to the capillary above the detector window door to secure the capillary Thread the free end of the capillary through the electrode thumbscrew The capillary should be as close to the electrode as possible without touching it The capillary should either be flush with the electrode or protrude beyond it no more than 0 5mm Affix a second small piece of thermal tape to the capillary above the electrode thumbscrew to secure the capillary The entire set up should look like this a le z Detector window Detector door e Close the heat plate door As a final step perform the Autosampler Calibration For a detailed tutorial please see the Autosampler Calibration document at http home appliedbiosystems com support apptech Page 5 of 7 Test CCD 4 Color and Seq Fill Capillary Module Applie
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