Automated Methods for PCR* Setup on the Biomek® FX
Lisa Knapp, Jason Fawcett, and Laura Pajak, Ph.D.
Beckman Coulter, Inc.
| Q. | Is any operator intervention required during the PCR setup protocol? |
| A. | No. Once the reagents and labware are placed on the deck, the protocol runs with no operator intervention. The operator must manually place the reaction plates into the thermal cycler once the protocol is complete. |
| Q. | Are there any special PCR reagents needed for this protocol? |
| A. | No. SureStart* Taq DNA polymerase from Stratagene was used for the protocol testing. Additionally, a single reaction using Taq DNA polymerase from Roche Biochemicals was performed using this protocol. No difference was seen between these two types of Taq. Other manufacturers Taq DNA polymerase should work with this protocol but they have not been tested. |
| Q. | Can I modify the protocol to use more template DNA, primers or a different total reaction volume? |
| A. | Yes. These protocols are designed to allow customization by the individual operators. However, different template concentration/volumes, primer concentration/volumes or reaction volumes were not tested in the development of these methods. |
| Q. | Are the reagents stable while kept at room temperature on the Biomek FX? |
| A. | Check the manufacturer’s recommendations for reagent stability. In the current protocol, SureStart Taq DNA polymerase from Stratagene and Taq DNA polymerase from Roche Biochemicals were used and no issues with reagent stability were observed. |
| Q. | Is cross contamination an issue? |
| A. | The methods developed were designed to reduce the risk of cross contamination to a minimum. This was accomplished by optimizing the deck layout to avoid any potential of “flyover” contamination caused by used tips passing over reaction or source plates, using new, clean tips for every action that involves potential contamination of the reaction, specifically, the transfer of template DNA, and adding reagents in the order of master mix, primers, if separate, and finally, template DNA to reduce the exposure of tips and reagents to template DNA. |
| Q. | Do I have to run the same number of plates as the protocols specify? |
| A. | No. The protocols have the maximum number of plates that can be setup according to the specific method. Each protocol can be modified to accommodate any number of plates up to the maximum. The protocol used should be selected on the basis of which reagents will be added and if the template is in a source plate or if it has been aliquoted to the reaction plates. The instrument setup and the method should be modified to reflect any changes. |
| Q. | Can I use different brands of PCR and 96-well plates? |
| A. | Yes. Different brands of plates can be used for these methods. However, ensure that your labware has been defined properly. If it is necessary to stack plates, the stack offset definitions are critical to avoid tip crashing. It is recommended that a dry run of the method be performed to ensure that labware definitions and stack offsets are correctly defined. |
* 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.
