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> Nucleic Acid Prep &
Purification > Sequencing
Setup > Sequencing Setup
Biomek®
FX
Sequence Reaction Setup Using the Biomek® FX Automated Workstation
Monique Mason, Jason Fawcett, and Laura Pajak, Ph.D.
Beckman Coulter, Inc.
| Q. | Does exposure time on the deck affect sequencing quality? |
| A. | The methods all have a completion time less than 12 minutes so there is not enough time for fluid evaporation, DNA degradation from extended periods at room temperature or enzyme activity reduction from prolonged exposure to light. Therefore sequencing quality will not be adversely affected. |
| Q. | How much user intervention is required? |
| A. | User intervention is only required in the beginning to set up the deck prior to running the method and immediately afterward to place the sequencing reaction plates in the thermal cycler. |
| Q. | Can reagent quantities be changed? |
| A. | Yes. These methods were prepared with much flexibility for the individual user, although any volume changes must be manually modified in the method. Keep in mind that these methods were developed and optimized using the amounts and labware given in the applications bulletin. |
| Q. | Can the number of plates vary? |
| A. | Yes. The maximum number of plates was utilized for each method created. The user may decide to use less than this maximum number of plates for any particular method. However, the deck setup and method will need to be modified to accommodate these changes. |
| Q. | Can different sequencing kits be used? |
| A. | Yes. As long as the method is modified to accommodate any volume changes. Note that the only kit used in the development of this applications bulletin on the Biomek FX was the ABI Prism* Big Dye* Terminator Cycle Sequencing Ready Reaction kit. |
| Q. | Can you sequence other types of DNA besides plasmid such as BAC and bacterial genomic DNA? |
| A. | Yes. Different types of DNA may be sequenced using these methods. However, the user would need to refer to the specific kit manual being used for reaction setup and protocol requirements. |
| Q. | Is there a specific reason for the deck layout for the sequencing reaction? |
| A. | Yes. The layout has been specifically designed to reduce the risk of contamination from tips crossing over open plates. |
| Q. | What if I want to make less than a full 96 well plate sequencing reaction? |
| A. | If you have less than a full plate for sequencing, simply leave unused wells empty. |
| Q. | When aspirating the reagents I see bubbles in the tips and in the liquid after the dispense step. What can I do to eliminate the bubbles? |
| A. | Reducing the speed at which the Biomek FX dispenses should eliminate any bubble problems. |
| Q. | Is the cleanup method specific for the different types of electrophoresis? |
| A. | Yes. The post sequencing cleanup method for use on the ABI3700 requires the use of a salt free, exceptionally clean protocol such as the DyeEx 96 kit by QIAGEN or the Gel Filtration Blocks by Edge BioSystems. If a less stringent method is used the capillaries may become clogged and problems with indistinguishable peaks will occur. If the sequencing will be done on the ABI377 a more cost efficient, salt-based cleanup such as ethanol precipitation should work just fine. The QIAGEN and Edge BioSytems’ kits will also work for the ABI377. |
| Q. | Can plates be stored prior to PCR while waiting for the thermal cycler? |
| A. | Check with the manufacturer of the sequencing kit used for recommendations in this area if this is a necessary step. ABI stated that the Taq is inactive at colder temperatures and therefore suggests refrigerating or freezing as long as the plate is not frozen and thawed multiple times. For this experiment, plates were left at -20ºC for approximately 3-4 hours without deleterious effects. |
| Q. | When using halfBD, how do the reagent volumes change? |
| A. | The only change is in the master mix volume. An example of volumes for one 20µL reaction would be as follows: 4µL of master mix, 4µL of halfBD, 4µL of H O, 2µL of primer (3.2pmol) and 6µL of DNA template (41.6ng/µL) for a final concentration of 250 ng. |
| Q. | What was the ethanol procedure used for post sequencing cleanup for the ABI377? |
| A. | The following ethanol procedure was used from the Beckman CEQ dye terminator cycle sequencing kit protocol:
Other ethanol procedures may work but were not pursued in this study. |
* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.
