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Project Documentation & Protocols: Maize Gene Discovery Project: ESTs: Protocols: Cycle Sequencing

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EST Sequencing: Cycle Sequencing

General Description: The DNA has been purified and precipitated by isopropanol/ethanol precipitation. Dideoxy sequencing, utilizing BigDyeä, is used to obtain sufficient copies of the DNA insert in varying read lengths. The DNA cycle extension products are then purified of contaminants using isopropanol precipitation. This protocol outlines the procedures for cycle sequencing and purifying the extension products in preparation for reading of sequence on the MegaBaceä 1000 capillary machine.

 

Materials & Reagents:
Material / Reagent Vendor Stock #

BigDyeä Terminator v3.0 ready reaction cycle sequencing kit

ABI

N8050001

Forward and reverse primer (0.125-0.25 pmol/ml)

Operon Technologies

N/A

Autoclaved Deionized and distilled water (Autoclaved ddH2O)

N/A

N/A

Template DNA (QIAGENâ preps.).

N/A

N/A

Sterile reservoir

Matrix

8096

FinnpipetteÒ Multichannel Digital Pipetter (8-channel), 0.5-10L

PGC Scientific

716284-02

Fisher Vortex-Genie 2ä Mixer

Fisher Scientific

12-812

Jouan CR412 Centrifuge

N/A

N/A

Hydra 96ä microdispenser

Robbins Scientific Corp.

1029-40-3

MicroAmpâ 384-well reaction plate

Perkin-Elmer/ABI

4305505

MicroAmpâ Clear adhesive covers

Perkin-Elmer/ABI

4306311

GeneAmpÒ PCR System 9700

ABI

N8050001

96-well plate base

Marsh

AB-0563

Beckman CS-6R Centrifuge

N/A

N/A

Plate sealers

Edge Biosystems, Inc.

48461

Isopropanol (Final concentration of 65-75%)

N/A

N/A

 

Procedure

Preparing Cocktail

  1. Take out primer and BigDyeä to defrost. Note: Primer may be quick-thawed by putting tube in water, but BigDyeä should be thawed slowly at room temperature in the dark.
  2. Take out a sterile reservoir.
  3. Dispense the following volumes into a sterile reservoir according to the amount of DNA desired (Use these volumes as standard for 2 ml of DNA. If adding more DNA, subtract 400 ml of H2O for each 1 ml of DNA added and vice versa if subtracting DNA in 1ml increments):
  4. 800 m l ddH2O
    800 m l primer
    1600 ml BigDyeä

  5. Using 10 ml 8-multi-channel FinnpipetteÒ, set fill and dispense volume appropriate to DNA volume being added so as to equal a 10 ml reaction volume. Dispense cocktail into quadrants of 384 well-plate (depending on quadrants needed).
  6. Remove QIAGENâ preps containing template DNA from 4 °C.
  7. Vortex for approximately 10 seconds on high (set to touch).
  8. Centrifuge template blocks till rpm reaches 1000, then stop.

Adding QIAGENâ Prep. DNA to BigDyeä Cocktail (utilizing Hydraä 96 microdispenser)

  1. See general procedure for operation of Hydraä 96 microdispenser.
  2. Using a preset program, set Fill Volume (FV) and Dispense Volume (DV) to volume of DNA to be added for cycle sequencing (See Preparing Cocktail, above).
  3. Perform a dirty and clean wash (A dirty wash is performed by using the volume of ddH2O used for the last clean wash. A clean wash uses a fresh volume of ddH2O to wash the syringes).
  4. Label plates according to library/plate number on QIAGEN blocks and direction of primer extension (i.e. 949003 x).
  5. Place first QIAGENâ block in tray and fill appropriate volume of DNA.
  6. Place 384-well reaction plate on 384-well reaction plate positioner so there is a tight fit and insert into Hydraä 96.
  7. Dispense volume of DNA into quadrant 1.
  8. Perform a dirty and clean wash as described previously.
  9. Repeat steps 5-8 for remaining quadrants.
  10. Cover each 384-well reaction plate with MicroAmpâ film and spin in Jouan CR412 centrifuge up to 1000 rpm and stop to release air bubbles.
  11. If not proceeding immediately to Cycle Sequencer, keep completed 384-well reaction plates out of light.
  12. Run cycle sequencing on a GeneAmpÒ PCR System 9700 using the following parameters for a 10 ml reaction:
    • Denaturation: 96 ° C for 10 seconds.
    • Annealing: 50 ° C for 5 seconds.
    • Extension: 60 ° C for 4 minutes.
    • Repeat steps 1-3 45 times (approximately 3.25 hours).
    • 4 °C until ready to purify the extension products.

Precipitating Extension Products

  1. Dispense 25 ml isopropanol into each well of a 96-well reaction plate (four per 384 plate).
  2. Put each plate into a 96-well plate base.
  3. Check cycle sequenced 384-well plates for condensation and spin at 1000-2000 rpm if needed.
  4. Label with X or Y (Forward or Reverse extension) and library/plate number.
  5. Perform a dirty and clean wash as described previously.
  6. Place 384-well plate on 384-plate positioner and align starting with the 1st quadrant.
  7. Place positioner into Hydra 96ä and aspirate a volume of 15 ml from cycle sequenced DNA in order to get all 10 ml in each well.
  8. Remove positioner and place appropriate 96-well plate with isopropanol into Hydra 96ä and press wash to mix DNA with isopropanol.
  9. Remove and place out of light.
  10. Repeat steps 5-9 for remaining quadrants.
  11. Spin 96-well plates with isopropanol and DNA at 4 °C for 30 minutes at 4000 rpm.
  12. After spin, immediately reverse spin 96-well plates by inverting on paper towels in Beckman GS-6R centrifuge. Let spin to 400 rpm and then quickly turn off to remove excess isopropanol and contaminants.
  13. Air-dry under fan for 10 . 15 minutes.
  14. Place clear adhesive tape over plates, crease over edges and attach sequencing bar-code label.
  15. Store plates up to a week in .20 °C freezer until ready for loading onto MegaBACEä 1000 machines.


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