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Detailed protocol for Snapshot

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

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

Snapshot reaction

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

Incubate using the following program

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

Post Snapshot clean up

  1. 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

  1. 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

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

 

Alison Brown December 28, 2001