Templiphi amplification of plasmid DNA

Purpose: Amplification of DNA for later sequencing

Original version by: Mike Alberti
Revision date: 9/12/06
Printed on: 9/12/2006
Background: This procedure is the preliminary step in the DNA sequencing procedures for the
Beckman CEQ 8000.

Materials

TempliPhi Amplification Kit (Amersham, Cat.# 25-6400-10 & 25-6400-50)

Procedure

1. Set out a sufficient number of 8-strip PCR tubes (0.2 ml tubes) and label them by number as
shown (try not to label the side of the tube as sweaty hands tend to rub the “permanent”
marker right off) or by date and/or a brief description across the tops of the tubes:

2. Add 2.5 µl of TempliPhi Sample Buffer (white cap) to the very top of each tube.

3. Gently tap (or briefly spin - preferred) the tubes against the table to settle the buffer to the
bottom.

4. Add 0.2-0.3 µl of E. coli glycerol stock from sample bank directly to buffer at bottom of
tube or for amplification directly from colonies, gently pick up a small amount of the colony
with a pipette tip and add directly to buffer at bottom of tube. For other starting templates
refer to the Templiphi manual.

5. Briefly spin contents to ensure the entire buffer-cell mixture has settled to the bottom of the
tube.

6. Place an adhesive plastic cover over the tops of the tubes and ensure tight seals.

7. Place the 8-strip tubes into the thermocycler and run the “95for3” protocol (95°C for 3
minutes followed by a 4°C hold) to lyse the bacterial cells and release the plasmid DNA.

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8. During incubation prepare a Master Mix consisting of TempliPhi Reaction Buffer (blue cap)
and TempliPhi Enzyme Mix (yellow cap). Add 2.5 µl reaction buffer to 0.1 µl enzyme for
each sample plus one additional sample to allow for pipetting error. For example:

If you had 48 samples:

49 x 2.5 µl = 122.5 µl Reaction Buffer
49 x 0.1 µl = 4.9 µl Enzyme Mix

9. Once the samples have cooled to 4°C, spin briefly and then remove the adhesive cover.
Next, add 2.5 µl of the Master Mix prepared in Step 8 to the top of each sample tube to
avoid contamination (NOTE: if up to 5 µl is accidentally added to each sample instead of
2.5 µl, the reaction will still work-continue on!).

10. Briefly spin contents to ensure the entire ~5 µl mixture has settled to the bottom of the tube.

11. Carefully place the 8-strip tubes into the thermocycler and apply 8-strip caps to the tops and
ensure seal by pushing down on the top with the bottom of a Sharpie marker or pen.

12. Run the “Amplify17hr” protocol (30°C amplification for 17 hours followed by enzyme
heat-inactivation at 65°C for 10 minutes and finally a 4°C hold).