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Home :: Technical Resources :: GeneReleaser® Protocols
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Technical Resources
GeneReleaser® General Protocol
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GeneReleaser® is a proprietary reagent which releases DNA from whole blood, cell cultures, bacterial colonies and the like. Lysis is accomplished directly in the amplification tube on a thermocycler. Typically, 1µl of specimen is lysed in a total volume of 20µl and amplification reagents are subsequently added to initiate amplification. GeneReleaser® sequesters cell lysis products which might inhibit polymerases and improves amplification yield and specificity. GeneReleaser® greatly simplifies the amplification of genomic DNA by avoiding the requirement to purify DNA. This avoids lengthy protocols and excessive sample manipulations which may introduce contamination.
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Materials and Equipment:
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Kit components:
1. GeneReleaser® is provided as a ready to use stock. There are 4 X 2ml tubes sufficient for 400 assays. Store at 4°C. DO NOT FREEZE!
2. A thermocycle profile is provided for the use of GeneReleaser®.
Other Equipment:
3. A programmable thermoycler and accessory reagents and equipment for amplification.
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General:
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It is assumed that the user is familiar with the programming and use of thermocyclers and the reactions which can be performed on them. No license to use PCR* is given or to be implied by this product or these instructions. A limited license for PCR can be obtained with the purchase of PCR reagents or thermocycling devices from Perkin Elmer.
Whole Blood Procdure:
1. Place 1µl of whole blood into the bottom of each thermocycling tube for each specimen.
2. Thoroughly resuspend the contents of the GeneReleaser® tube by inverting 10-20X.
3. Add 20µl of the resuspended GeneReleaser® to each tube.
4. Perform the thermocycle program described below.
5. Perform the amplification reaction according to your optimized protocol.
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Bacterial Colony Procedure:
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1. Place a single well isolated colony into the bottom of a thermocycle tube for each specimen.
2. Perform steps 2-5 as per these steps in the Whole Blood Procedure above.
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Tissue Culture Procedure:
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1. Place 1µl of cells at a density of 103-108 cells/ml into the bottom of a thermocycle tube for each specimen.
2. Perform steps 2-5 as per these steps in the Whole Blood Procedure above.
Thermocycle Program: NOTE: Reaction volumes and concentrations should be adjusted proportionally to allow for the 20µl GeneReleaser® volume. In some instances a new magnesium titration may need to be performed to assure optimum amplification. We suggest the range of 1.5mM - 4.0 mM Mg in 0.5mM increments for this titration.
Following the thermocycler manufacturer's procedure and using the default transition rates between temperatures, enter and run the following program:
Note: When using the Perkin Elmer GeneAmp® PCR system 9600 or 2400 all times below should be reduced by 50%.
Overlay the specimens with mineral oil.
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Step
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Temperature
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Heating Rate
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| 1. | 65°C | hold 30 sec. |
| 2. | 8°C | hold 30 sec. |
| 3. | 65°C | hold 90 sec. |
| 4. | 97°C | hold 180 sec. |
| 5. | 8°C | hold 60 sec. |
| 6. | 65°C | hold 180 sec. |
| 7. | 97°C | hold 60 sec. |
| 8. | 65°C | hold 60 sec. |
| 9. | 80°C | hold. |
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| 10. | Adjust temperature as necessary. |
| 11. | Add amplification reagents without vortexing or otherwise mixing the amplification tube contents. |
| 12. | Begin your optimized amplification. NOTE: It is very important that the very first denaturing step of the first cycle be at 94°C for 2-5 minutes depending on brand of cycler, reaction volumes etc. |
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Performance Characteristics:
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GeneReleaser® is designed for release of genomic DNA. If low copy number DNA is being processed i.e. virally infected cells, then cellular enrichment should be performed.
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