Pharmacogenetics of Asthma Treatment

Detailed protocol for Snapshot

After single or multiplex PCR, the PCR product must be cleaned up using EXOSAP-IT

2. Add 2 m l EXOSAP-IT to 5 m l PCR product
3. Incubate at 37^oC for 1 hour, then 80^oC for 30 minutes.

Snapshot reaction

2. Make a pool of the Snapshot internal extension primers to a final concentration of 0.8 m M of each primer.
3. 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.
4. To increase the intensity of extended product peaks, increase the concentration of the extension primer in the pool.

Incubate using the following program

2. 96^oC 10 seconds
3. 50^oC 5 seconds
4. 60^oC 30 seconds
5. Return to Step 1 for 25 more cycles
6. 4^oC Hold

Post Snapshot clean up

2. Add 2 m l Shrimp Alkaline Phosphatase diluted 1:2 with SAP dilution buffer, to each 10 m l sample and incubate at 37^oC for 90 minutes.

Sample preparation for running on an ABI 3100

2. Add 9 m l HiDi Formamide to 0.5 m l LIZ size standard and 0.5 m l cleaned SnaPshot reaction. Denature at 95^oC and quench on ice for 2 minutes minimum.

ABI 3100 running conditions

2. Run Module SNP36_POP4DefaultModule
3. Analysis Module GS120Analysis.gsp
4. Polymer POP4 (4316355, Applied Biosystems)
5. 36 cm Capillaries (4315931, Applied Biosystems)

Alison Brown December 28, 2001