FAQ: Can the phi29-XT RCA Kit be used to amplify large, circular DNA constructs such as BACs and Fosmids?

Yes, the phi29-XT RCA Kit can be used to amplify large, circular DNA constructs. If using purified BAC or Fosmid DNA as starting material, follow the protocol in the manual: phi29-XT RCA with purified circular DNA input. For amplification of BAC or Fosmid DNA directly from a bacterial colony, please follow the protocol in Section II with the following modifications:

  1. DNA denaturation: resuspend a single colony in 5 µL denaturation buffer (60 mM KOH, 2 mM EDTA), and lyse the cells/denature DNA at 60°C for 10 minutes. Immediately place on ice. 
  2. Neutralize the denatured DNA with 5 µL neutralization buffer (75 mM, Tris-HCl, pH 7.0).
  3. To improve the specificity of BAC/Fosmid DNA amplification, exonuclease-resistant site-specific primer pairs (0.2 -1 µM) are highly recommended to replace the random primers. 
  4. Increase the RCA time to 4-8 hours.