The system is designed to robustly and reliably amplify DNA under pre-optimized standard reaction conditions. GE Healthcare claims that the kit contains high-purity reagents for end-point PCR reactions to ensure the lowest possibility of pro- and eukaryotic DNA contamination. Little information is given about the characteristics of the enzyme, but by contacting their technical service, I found out that the Taq lacks proof-reading abilities. In addition, the protocol booklet, which is included with the kit, can be downloaded from GE Healthcare’s website. This booklet contains a brief overview of PCR, recommendations for primer design, a protocol for setting up PCR reactions, as well as a section how to avoid nucleic acid contamination and a troubleshooting guide.
Kits are provided as either single tubes (thin-wall 0.2 ml or 0.5 ml) or 96-well plates; the plates can be easily divided into 8-well stripes so that the desired number of reactions can be run. Since the beads are lyophilized, the kits can be stored at room temperature. The beads contain the reagents necessary to perform pre-optimized, standard PCR in a 25 µl reaction volume and contain the following reagents per tube: approximately 2.5 units PuReTaq polymerase, 200 µM dNTP (dATP, dCTP, dGTP, dTTP), BSA, stabilizers and reaction buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl2). Additional MgCl2 can be added to the reaction, if necessary; to save you the calculations for determining how much of your MgCl2 stock to add, a table is the included in Appendix 3 of the booklet.
To set up the PCR reaction, the user only has to add the specific primers, dH2O and DNA. Making a master mix of the dH2O and primers (and additional MgCl2, if necessary) is recommended; generally 23-24 µl (depending on the volume of DNA) of this mix will be added to each tube. The template DNA is then added last in order to avoid cross-contamination. The recommended amount of DNA (50 pg for plasmid DNA or 50 ng for genomic DNA) is added to bring the final volume to 25 µl. (Note: These amounts are offered as guidelines; the optimal amount of DNA should be determined empirically.) Gentle mixing of the reaction is recommended to fully resuspend the beads (the solution should be clear) after sealing the tube with the cap tightly (make sure you hear a snap sound). Make sure that the beads have fully resuspended as I found that the DNA either did not or poorly amplified when the beads were not completely solubilized. After mixing, it is also important that the tubes are centrifuged to collect the reaction at the bottom of the tube before placing them in the thermal cycler.
The PuReTaq Ready-To-Go™ PCR system is excellent for less experienced lab personal and/or high throughput PCR amplifications (e.g. screening tail-DNA from mouse colonies). It limits sequentially pipetting of small volumes. Since the amplification of DNA with this system is robust and reliable over a wide range of genomic DNA concentrations (30-100 ng of DNA), the DNA/primer concentrations for individual amplifications had to be established only once. Further reactions were set up with the same volume of DNA obtained from the purification without determining the DNA concentrations. This PCR system is less suitable for more challenging reactions like amplification of single copies. The PCR kit is very robust and reliable, easy to use with multiple samples and cost efficient (< $1.60/reaction).