New England Biolabs, E1604S, phi29-XT WGA Kit

Related Categories Isothermal Amplification & Strand Displacement Applications Whole Genome Amplification,, Isothermal Amplification FAQ Q: How can I increase the WGA yield when using the phi29-XT WGA Kit (NEB #E1604)? A: Higher WGA product yield may be obtained by scaling up the reaction volume, increasing the incubation time, or increasing the amount of input DNA. Please note that increasing the incubation time may lead to more amplification of non-specific products in the absence of template. Q: At what temperature range can the phi29-XT WGA Kit (NEB #E1604) 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 or higher than 43°C. 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). Q: Is the phi29-XT WGA Kit (NEB #E1604) suitable for amplification of short DNA fragments? A: The phi29-XT WGA Kit uses Multiple Displacement Amplification (MDA), which is more suitable for long DNA fragments, as the amplification efficiency diminishes when the template length of the starting material decreases. Q: When using the phi29-XT WGA Kit (NEB #E1604) do I need to purify the WGA products prior to downstream applications? A: Typically, WGA products can be directly used in downstream applications without cleanup. Many downstream applications only require dilution of the WGA products before use. If purification is necessary, the WGA products may be cleaned up using 0.6X SPRI® beads, following manufacturer’s recommendations. Q: How do I quantify my WGA products generated using the phi29-XT WGA Kit (NEB #E1604)? A: WGA 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 WGA products can be quantitated by measuring the absorbance at 260 nm or by NanoDrop®. Q: When using the phi29-XT WGA Kit (NEB #E1604) can the reaction volume be scaled up or scaled down? A: Yes, the reaction volume can be scaled down to 10 µl and scaled up to 50 µl from the recommended 20 µl. Further scaling may be possible, but it has not been tested. Q: Is the phi29-XT WGA Kit (NEB #E1604) suitable for single cell DNA amplification? A: Yes, the phi29-XT WGA Kit is recommended for amplification of single cell DNA. For cells with thick cell walls (e.g., plant cells), cell lysis may be necessary to release the DNA prior to the denaturation step. Q: Do I need to extract DNA from whole cells prior to using the phi29-XT WGA Kit (NEB #E1604)? A: For cells that are easily lysed, the DNA denaturation step included in the protocol for the phi29-XT WGA Kit is sufficient. However, for cells that are difficult to lyse, DNA extraction may be beneficial. The Monarch® Spin gDNA Extraction Kit (NEB #T3010) can be used to extract DNA from a broad range of sample types, such as cells, blood, tissue, bacteria, yeast, saliva, and swabs.