AmpliCycle Sequencing

Dominik Haudenschild, Monday, October 06, 1997

1: Into separate tubes labeled G,A,T,C aliquot 2µl of appropriate termination mix

2: Reaction mix

Water to 30µl total volume

Primer 20pmol 1µl of 1:5 dilution of 100pmol/µl

a ³³P dCTP 10µCi/µl 0.5µl to 1.0µl

Template 10 to 100fmol 1µg of 4361bp plasmid is 350 fmol

10x Cycling mix 4µl

Primers:________________________________________________________

Templates:_____________________________________________________

________________________________________________________________

3: PCR

Dispense 6µl of reaction mix into each tube G, A, T, and C

4: Insert tubes into PCR machine preheated to 95°C

5: Run program

1. Initial Denature 95°C for 1:00

2. Denature 95°C for 0:15

3. Anneal __ °C for 0:30 (t_m minus ~3°C)

4. Extend 72°C for 1:00

5. Goto step 2 24 additional cycles

6. Hold 4°C for 45:00 at most

6. Stop reaction by adding 4µl Stop solution

7. Store samples frozen until ready to run

8. Heat samples to 90°C for 3 minutes just prior to loading

9. Load 3µl of sample per lane