Gap Closure using the Amersham Phi29 Sequence Finishing Kits for DNA Amplification Prior to Sequencing

version dated 05-26-05

(adapted for use in the Roe lab by Shaoping Lin)

1. Mix 1ul of DNA template (plasmid template) with 4ul of sample buffer (white cap tube in the Amersham Sequence Finishing Kit,#25-6401-01A). Denature by heating to 95⁰C for 3 min. Cool to 4⁰C.

2. Defrost the reaction buffer (blue cap tube in the Amersham Sequence Finishing Kit #25-6401-01B) and enzyme mix (yellow cap tube in the Amersham Sequence Finishing Kit, #25-6401-01C) by placing on ice just prior to use. Gently mix 4.75ul reaction buffer with 0.25ul enzyme mix and then add 5ul of this mixture into the cooled sample from the step 1.

3. Mix it gently by pipitting up and down using an Eppendorf pipette and then centrifuge the plate at 1000 rpm for 2 seconds to concentrate the mixture to the bottom of the wells in 96 well plate.

4. Incubate samples at 10⁰C for 18 hours at thermal cycler.

5. Heat the sample to 65⁰C for 10 minutes to inactivate the enzyme after step 4.

6. Store the samples at -20⁰C if you desire.

7. Use 2ul or 4ul of this Phi29 amplifiied DNA in a standard DNA sequencing reaction.

Note: The Phi29 reaction and sample buffer, enzyme mix should keep on ice. Do not allow the mixture to warm above 4⁰C prior to the amplification reaction.