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Project Documentation & Protocols: Maize Gene Discovery Project: ESTs: Protocols: Loading DNA Samples

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EST Sequencing: Loading DNA Samples

General Description: The insert DNA has been cycle sequenced using the dideoxy method and purified by isopropanol precipitation. The purified cycle product is then run through capillary array electrophoresis on the MegaBACE(tm) 1000. This protocol outlines the procedures for prepping the purified DNA and loading the DNA sample on the MegaBACE(tm) 1000 for sequence analysis.

 

Materials & Reagents

Material / Reagent

Vendor

Stock #

MegaBACEä Long Read Matrix

Amersham Pharmacia Biotech, Inc.

US79676

1X MegaBACEä LPA Buffer (prepared from 10X stock solution)

Amersham Pharmacia Biotech, Inc.

93-79677

MegaBACEä Loading Solution w/ formamide

Amersham Pharmacia Biotech, Inc.

US79676

96-well polypropylene CyclePlateâ

Robbins Scientific Corp.

1055-90-0

Pressurized Nitrogen

N/A

N/A

Purified DNA sample plate

See Cycle Sequencing Protocol

N/A

Fisher Vortex-Genie 2ä Mixer

Fisher Scientific

12-812

Deionized water

Prepared at Genome Center

N/A

Eppendorf 5415C centrifuge

N/A

N/A

Jouan CR412 centrifuge

N/A

N/A

Plate sealers

Edge Biosystems, Inc.

48461

 

Procedure

Matrix Injection

  1. Initiate Host Scan Controller and then start Instrument Control Manager (ICM) programs.
  2. Retrieve 6 tubes of Long Read Matrix from fridge per MegaBACEä 1000 and spin at 4000 rpm for 4 minutes in an Eppendorf 5415C centrifuge.
  3. Fill each well in a 96-well cycle plate with 200 ml 1X LPA loading buffer and mark to differentiate from DNA sample (one plate per MegaBACEä 1000).
  4. If this will be the first run of the day, run a tip rinse on the machine(s) with deionized water.
  5. Turn on high-pressure to nitrogen before proceeding with matrix loading and occasionally check this reading within Instrument Control Studio (ICS). Note: high pressure should be between 950 and 1050 psi; low pressure should read "normal."
  6. Start Matrix Fill and Pre-run and follow instructions on machine(s).
  7. Load buffer plate and unscrew matrix tubes while script is running.
  8. When prompted, add matrix tubes and proceed to DNA sample preparation.

DNA Sample Preparation

  1. Add 8 ml of formamide loading solution to each plate to be run.
  2. Stack plates on vortex by tying rubber bands to secure them and shake on vortex power 4 for 4 minutes.
  3. Spin again briefly at 1000 rpm.

DNA Sample Injection

  1. During Pre-run, begin plate setup.
  2. Open MB1000 folder.
  3. Open Plate Setup.
  4. Enter your initials and the plate ID information on the screen display.
  5. Check to ensure plate ID and plate number match those on plate label. Check again.
  6. Click Setup Plate.
  7. Once samples have been logged into the database, hit quit and proceed to Plate Setup on Instrument Control Manager.
  8. Hit new button to enter new plate ID.
  9. Enter plate ID and appropriate injection parameters (injection voltage and time) for each MegaBACEä 1000. Slight optimization is required in order to find proper loading conditions. In general, 30-40 kilovolts (kV) * seconds works well as a starting point with EST samples.
  10. Click save to save new parameters for sequencing run.
  11. Initiate Inject and Run Samples and follow instructions on machine(s).
  12. The machine will prompt for action with the following messages:
    • Load full water tank and save buffer plate (deionized water)
    • Quickly load sample plate
    • Quickly load water tank
    • Load saved buffer plate
  13. Since sample loading is time sensitive, perform steps in an efficient, timely manner.
  14. Go to Run Image on Instrument Control Manager (ICM) to view signals on each capillary.
  15. After 30 minutes, check current monitor to be sure there is no clogging.
    • If there are 0.s and no peaks are occurring, clogging is occurring in those capillaries.
    • If enough capillaries exhibit this, stop and re-do the injection, varying either the injection parameters (voltage and time) or initial DNA concentration as determined during Cycle Sequencing (see Cycle Sequencing protocol).
  16. After run is complete, wait for base calling to be completed. If desired, check MegaBACE Scorecard for read length sequence success and/or FTP sequence results by going to the MDFileman application.
  17. To put machines to sleep for up to 3 days (60 hours), initiate Store Capillaries on Instrument Control Manager and follow instructions to specify amount of time.


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