Phase 

Sequencing Protocol

After clones have been plamid-purified (using Qiagen Qiaprep kits), plasmid DNA is sequenced using the Amersham DyeNamic Sequencing Kit. The following protocol is used: 

(Adapted from Amersham DyeNamic Sequencing Kit protocol)

1. To a 1.7 ml tube add 4 ul plasmid prep, 4 ul Sequencing reagent premix, 2 ul M13 F (5 pmol) universal sequencing primer.

2. Sequencing reactions are then thermal cycled according to the following reaction:

3. Denature: 95 ^o C for 20s

   Anneal at 50 ^o C for 15s       X 25 cycles

   Extend at 60 ^o C for 60s

4. Clean up sequencing reactions by pelleting with 95 % EtOH and 250mM EDTA, followed by washing pellet with 70% EtOH.

5. Pellets are dried, resuspended then run on a ABI377XL automated sequencer.

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