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Detailed protocol for Snapshot
After single or multiplex PCR, the PCR product must be cleaned up using EXOSAP-IT
- Add 2 m
l EXOSAP-IT to 5 m
l PCR product
- Incubate at 37oC for 1 hour, then 80oC for 30 minutes.
- Make a pool of the Snapshot internal extension primers to a final concentration of 0.8 m
M of each primer.
- To 3 m
l EXOSAP'ed PCR product add 2.5 m
l Snapshot Multiplex reaction mix, 2.5 m
l HalfBD, 1 m
l of pooled primer and make up to 10 m
l with water.
- To increase the intensity of extended product peaks, increase the concentration of the extension primer in the pool.
Incubate using the following program
- 96oC 10 seconds
- 50oC 5 seconds
- 60oC 30 seconds
- Return to Step 1 for 25 more cycles
- 4oC Hold
Post Snapshot clean up
Add 2 m
l Shrimp Alkaline Phosphatase diluted 1:2 with SAP dilution buffer, to each 10 m
l sample and incubate at 37oC for 90 minutes.
Sample preparation for running on an ABI 3100
Add 9 m
l HiDi Formamide to 0.5 m
l LIZ size standard and 0.5 m
l cleaned SnaPshot reaction. Denature at 95oC and quench on ice for 2 minutes minimum.
ABI 3100 running conditions
Run Module SNP36_POP4DefaultModule
Analysis Module GS120Analysis.gsp
Polymer POP4 (4316355, Applied Biosystems)
36 cm Capillaries (4315931, Applied Biosystems)
Alison Brown December 28, 2001