New England Biolabs, E1603L, phi29-XT RCA Kit

Related Categories Isothermal Amplification & Strand Displacement Applications Isothermal Amplification FAQ Q: What is the advantage of using phi29-XT DNA Polymerase instead of wild-type phi29? A: Generally, phi29-XT DNA Polymerase, under optimal conditions, generates more product in less time than the wild-type enzyme. Additionally, phi29-XT is more sensitive than the wild-type phi29, making it ideal when the input DNA amount is extremely low (i.e., femtogram of circular DNA). These qualities make phi29-XT the ideal choice for most RCA applications. Wild-type phi29 phi29-XT Fast (3-12 kb/min) ✓ ✓ Highly Processive ✓ ✓ High Fidelity ✓ ✓ Strand Displacement ✓ ✓ Optimal Temperature 30°C 42°C Yield ✓ ✓✓ Sensitivity ✓ ✓✓ Q: How can I increase the RCA yield? A: Higher RCA product yield may be obtained by increasing the incubation time or the amount of input DNA. Please note that increasing the incubation time may lead to more amplification of nonspecific products in the absence of template. Q: At what temperature range can the phi29-XT RCA Kit be used? A: The optimal reaction temperature range is 40 to 42°C. Amplification using this kit is not recommended at temperatures below 37°C. Q: Can the phi29-XT RCA Kit be used to generate single-stranded DNA? A: Yes, long, single-stranded DNA can be generated by replacing the Exonuclease-Resistant Random Primers with one or more unidirectional primers (0.2-1 µM final concentration). Q: Can the phi29-XT RCA Kit be used to amplify large, circular DNA constructs such as BACs and Fosmids? A: 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: 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. Neutralize the denatured DNA with 5 µL neutralization buffer (75 mM, Tris-HCl, pH 7.0). 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. Increase the RCA time to 4-8 hours. Q: Does the input DNA template have to be circular? A: Rolling circle amplification does require circular template DNA. However, the random primers included in this kit can also anneal to linear DNA, and phi29-XT will amplify the linear DNA via a Multiple Displacement Amplification (MDA) mechanism. Q: How can I verify successful amplification and specificity of my RCA reaction? A: RCA product quality and specificity can be assessed by digesting with a restriction enzyme and running the digested products on an agarose gel beside a DNA ladder such as Quick-Load® Purple 1 kb Plus DNA Ladder (NEB #N0550). Digested RCA products should show the same fragment sizes expected for the digested plasmid DNA template. Q: How can I debranch my RCA products? A: Please refer to the manual for general debranching instructions. Q: Do I need to purify the RCA products prior to downstream applications? A: Many downstream applications only require dilution of the RCA products before use. If purification is necessary, the RCA products may be cleaned up using 0.6X SPRI® beads, following manufacturer’s recommendations. Q: How do I quantify my RCA products? A: RCA products may be directly quantified using the Quant-iT® PicoGreen® dsDNA Assay Kit or with a Qubit® Fluorometer after dilution to the appropriate detection range. Alternatively, purified RCA products can be quantitated by measuring the absorbance at 260 nm or by NanoDrop®. Q: Can phi29-XT DNA Polymerase be heat inactivated? A: Yes, phi29-XT can be heat inactivated by incubation at 65°C for 10 minutes. Q: Can I use this kit to incorporate 5-methyl-dCTP? A: Yes, the phi29-XT RCA Kit can be used to incorporate 5-methyl-dCTP by replacing up to 100% of the dCTP with 5-methyl-dCTP (NEB #N0356). Q: Does phi29-XT DNA Polymerase work in wild-type phi29 DNA Polymerase Reaction Buffer? A: Yes, phi29-XT does amplify in 1X wild-type phi29 DNA Polymerase reaction buffer (NEB #B0269). However, the yield is typically diminished compared to synthesis in 1X phi29-XT Reaction Buffer (NEB #B0572). Q: Can phi29-XT DNA Polymerase be used in wild-type phi29 RCA reaction conditions (1X phi29 DNA Polymerase Reaction Buffer at 30°C)? A: Yes, phi29-XT DNA Polymerase will work in the wild-type phi29 reaction conditions and it will generate comparable yield to the wild-type enzyme under these conditions. However, for phi29-XT DNA Polymerase, higher yields are expected when carrying out the reaction at 42°C. Q: Use of specific primers for RCA with phi29-XT A: Based on limited observations, primers of 13-16 bases are more efficient than longer primers. As a general primer design rule (RCA, PCR, etc.), try to avoid more than 3 Gs or Cs in the last five bases to avoid mis-priming. It is critical that the 3' end is protected from the strong exonuclease activity of phi29 by including 2-3 phosphorothioate bonds. Q: What is the difference between the phi29-XT WGA Kit (NEB #E1604) and the phi29-XT RCA Kit (NEB #E1603)? A: phi29-XT DNA Polymerase is efficient at amplifying circular DNA by Rolling Circle Amplification (RCA) as well as genomic DNA by Multiple Displacement Amplification (MDA). However, the phi29-XT polymerase included in the phi29-XT WGA Kit (NEB #E1604) has been formulated to reduce GC bias during amplification of human genomic DNA. For RCA, we suggest using the phi29-XT RCA Kit (NEB #E1603).