Phase  

CEQ Sequencing Checklist For Quarter Reactions Using the 570F Primer & Sequencing Buffer

 Details  

• Mix DNA sample and MilliQ in wells of the 96 well plate. Depending on the concentration of the sample, MQ should be added to make up a total template plus H2O volume of about 11µl. The addition of 570F primer 15uM (1µl), CEQ DTCS Master Mix(2.0µl), and sequencing buffer (1.0µl) bring the total reaction volume to 15 µl. Appropriate amounts of MilliQ and template should be added based on template concentration from Pico green quantification. Target amount of DNA per reaction is 165ng!!
  For example:

• Mix MM, Primer, and Sequencing Buffer together separately from diluted DNA sample. Keep on Ice!!! Volumes: 1.0ml of Primer plus 2 ml premixed MM and 1.0µl of Seq. Buffer are added to each sample.
  Account for pipetting error by preparing extra MM/Primer/Buffer Solution
• Cover the plate with plastic cap strips and run DNA samples at 86°C for 5 min to allow plasmid + insert to denature.
• Add 4.5 ml of Primer/MM/Buffer solution to each well=>cover with plastic cap strips=> run PCR/sequencing cycle: 45cycles of 96°C for 20sec. =>50°C for 20 sec.=>60°C for 4min. Followed by an infinite hold at 4°C.

Plate Precipitation

• Spin samples down and add 5 ml of stop solution to each sample. Not Done On Ice!
  NOTE: allow for pipetting error and prepare for 14 additional samples!
  Mix by hitting plate against palm of hand.

• Add 60 ml of -20°C 95% EtOH to each well and cover plate with Cap Strips=> Mix by hitting plate against palm of hand.
• Place plate in centrifuge at 6102x g for 35 min at 4°C
• Invert plate onto paper towels and let EtOH come out.   Turn plate right side up again and spin at 6102x g for a few min. to secure pellet.
• Remove plate=>place folded paper towel on plate holder =>Spin in inverted position at 300 rpm for 20 sec.
• Rinse the pellets 2 times with 150 ml -20°C 70% EtOH=>Spin plate after covering it up with Al tape at 6102 x g for 15 min at 4°C after each wash=>Invert plate on paper towels to remove EtOH turn right-side up and spin at 6102x g for a few min=>add paper towels to plate holder before doing the inverted spin at 300rpm for 20 sec.
  ***Do not vortex the samples after the addition of the 70% EtOH***
• Allow pellets to dry by placing the plate near the edge of a hood and lowering the sash to create substantial airflow over the wells. Allow pellets to dry for 10 min.
• Resuspend each pellet by adding 40 ml of Formamide.  Tapping the plate against the palm of your hand should agitate the samples enough to resuspend them.
• Spin plate so that no formamide exists on the sides of the well and add 1 drop of mineral oil on top of each sample. (Samples are kept in the 4°C fridge until sequencing starts.)

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Create a sample sheet

• Turn on CEQ first
• Turn on the Computer => There is no password so click OK
• Click on CEQ icon => Data Manager => Caron Lab=> File=> Set as working database.
• Highlight Caron Lab=>Right click=>New Project=>Rename=> (ex)101602_ENDNA76 1-96
• Name the samples and select the method you want them to be run with.  Note: The LFR methods only work with the 33-75B short capillary arrays.
  To name the samples: Click on the Sample Setup icon=> Start=> Programs=> Excel=> type in sample names=> Copy & Paste to sample sheet
• Select an appropriate project for your samples, i.e. Denature temp & time, Injection voltage & time, and Separation voltage & time. For the current work use: Denature 90 C for 120 sec, Inject 2.0 KV for 20 sec, and Separate 4.2 KV for 110 min.
• Check to make sure that the Automatic Analysis option is not selected.
• Save the sample sheet by clicking => Save: Save as=> Go to “project name” to find    name=> Instead of default select sample name i.e.101602_Run=> OK=>Check in data manager under Sample Plates to see if saved.

Prepare the instrument for a run

• Sample setup: Go to New=> Data Manager from the top panel menu (Make sure the right Working Database is selected), Hit Run (from top panel and should say “no capillaries”) => OK
• Load Capillaries:  (If capillary needs to be changed) 
  • Click Replenish=>Release Manifold plug
  • Unlatch the black hood by pulling the two rubber latches
  • Remove the Manifold access door by unscrewing the two screws
  • Remove the Capillary access door (Plenum) by unscrewing the two screws
  • Replace MQ water in rinsing dish (might contain some acrylamide)
  • Remove the Manifold Plug (leave in CEQ area).
  • Remove new capillaries and take of cover carefully (wiggle gently from side to side)
  • Rest uncovered capillaries in wetting tray (Be careful not to bend or cross the capillaries while trying to position the capillaries in the wetting tray)
  • Take off cover from capillary window and wipe of gently 2-3 times with wetted foam Q-Tip (with MQ & wipe unidirectional)
  • Take off plastic tip of the manifold plug and insert onto the pins & lock in
  • Take the capillaries out of the water dish and guide them into pins on back of manifold access door
  • Replace the access manifold door
  • Replace the capillary access door (Plenum)
  • Lead capillaries through groove on side of capillary access door
  • Replace block hood
  • Shut Lid
  • Fill in Serial number
  • The OK button should pop up on screen=> hit OK, or select “DONE” (Exposure of capillaries to air should be kept to minimum!) Now the capillaries should automatically be fed into washing dish.
  **In the Install Capillary Array dialog box, select the proper part number for the array and enter the serial number.  If this is a new array: be sure to click the Set to New button.  Click Done.  Important Note: new 33-75B capillary arrays need to be conditioned before use.  To do this, run a column of 8 wells of only SLS (Formamide) & Mineral oil with the Condition method.**
  Note: the covers from the capillaries contain acrlymide– make sure to put them back in the plastic box and then in the fridge. The solution might solidify if left out.
• Load Cartridge: Select Replenish=>Release Gel Cartridge.  If this selection is not highlighted then the Install Gel Cartridge option will be highlighted indicating that the gel plug is in place. Open the gel access door and pull the lever on the gel assembly towards you.  The program will yell at you and reset but this is ok.  The gel plug should be an expired gel cartridge that is marked to indicate that it is a gel plug. (If the gel plug is in place, remove it and install a gel cartridge.  If it is not the gel plug, you will need to check how long the installed cartridge has been on the instrument.  Look at the Life tab (middle left of Run module screen) and confirm that the cartridge has not been on the instrument longer than 3 days.  If it has, discard it and get a fresh one from the 4°C fridge.)
• Select Replenish=> Install Gel Cartridge.  In the dialog box, enter the lot number of the new gel cartridge (Expiration date), the Part # (608010), and click the Set to New button to reset the timer.  Click Done.
• Perform a capillary fill to remove any bubbles that were introduced: click Direct Control=>Gel Capillary Fill=>Fill.  To observe what is going on click Run Diagnostics=>Status, this checks the pressure, which should be going up to about 800 PSI, also you can click on “event window” for more information.
• Perform an optical alignment by selecting Direct Control=>Optical Alignment=> Align.
• Check the background of the array by selecting Run=>Monitor Baseline.  Click the Enable Monitor baseline checkbox and click OK.  With the Data Monitor view selected in the main window of the Run screen, check the background signal levels of the eight capillaries.  If any of the capillaries have a signal level >6000 counts, the array read window should be cleaned. 
  (To clean the window you will need to click Replenish=>Release Capillary Array, pull out the manifold portion of the array and wipe the window on both sides, in one direction, with a foam swab moistened with a drop of DI water.  If this does not bring the background down, then it is possible that the array has expired or been used for >100 runs or it can be that there are still bubbles in the array and another manifold purge must be done.)
  Approximately 150 runs (plates) are possible with one array. If values are too high: select run=>manifold baseline=>disable. Then select run=>manifold purge and wait till window prompts and check baseline once more.
• Load plates: Prepare buffer plate and get samples from live fridge. Each sample needs to have a corresponding well in buffer tray that is filled 3/4 with Sequencing buffer solution (live fridge).

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Start your run

• A run can be started from the Run drop-down menu in either the Run Module or the Sample Setup module.
  A:  From the Sample Setup module, select Run=>Start.  A Confirm Configuration window will pop up to confirm that there is either formamide or a sample in the positions indicated by the sample sheet.
  B:  From the Run module, select Run=>Start Sample Plate.  Select the saved sample sheet that you wish to run. A Confirm Configuration window will pop up to confirm that there is either formamide or a sample in the positions indicated by the sample sheet. 
  ** Important Note: Formamide needs to be in all empty sample plate wells and buffer needs to be in all selected buffer plate wells, do not run the capillaries dry or in water.
• Click Load Plates.  Click Unload to open the access cover.  Put in your buffer and samples plates – cover buffer plate with “sled”.  Remove the wetting tray, wash it out, and fill it to the line with DI water before putting it back into the machine. 
• Click Load.
• When the Confirm Configuration dialog box returns, the Start Button will now be highlighted.  Click Start to begin the run.

Analysis & Saving of Data

• Analysis:
  After the run is complete:  Select Sequence Analysis=> Analysis=> Batch Analysis=> Project Name=> Select our Sample & Highlight all the files=> Select the > key to load=> OK=> Default Sequence Analysis=> OK        
• Saving:
  A CD is used to save all the data from the run to be analyzed later.  The data are saved as SCF & Text files.  So far formatting the CDs that we have is not necessary.
  Start=> Programs=> Adaptec Easy CD=> Create CD=> Data=> Data CD
  Go back to the database=> open the run=> Sequence Results=> highlight all samples=> right click=> Export=> SEQ Output=> check Result Output box=> right click=> Export=> SCF 3.0=> check Raw Data & Result Data=> Save to a folder
  *Don’t need the change file name, just save as default.
  Go to the CD to find files=> Highlight the saved files=> Click on the Add button=> Create CD
• Back up & Removal of Data:
  Back up all stored data from the sequencer onto a CD by exporting and saving the sequence results and CQ files and SCF files.  After the CD is burned and has been checked to verify if the data is present on the CD, remove the project from the database.  Once you have completed your work to that particular database and have saved the information to CDs from each project, you need to delete the database.  Leaving the database open takes a lot of memory, even though individual projects have been removed from it.  Deleting the database opens up used memory and allows the system to run faster.       

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