Frequently Asked Questions
1. What are the advantages of TempliPhi™ over conventional DNA sample processing methods, culture and workflows?
The biggest advantages are the elimination of plasmid miniprep, potential elimination of overnight culturing step, and the normalization of DNA yield. In addition, there are significant savings on consumables, waste disposal costs, hands-on time and overall time of template preparation for DNA sequencing. There is no need to purify amplified DNA by any method before DNA sequencing. Amplified DNA can be directly used for sequencing.
2. Can the random hexamers used in TempliPhi interfere with sequencing reactions?
No. The Tm of the hexamers used in TempliPhi is far below the typical cycle sequencing temperature ranges.
3. Can TempliPhi DNA “overload” the MegaBACE sample injection?
Yes. Too much of any DNA can potentially overload MegaBACE capillaries. Sample-to-sample DNA concentration variability can potentially be reduced with TempliPhi by allowing reactions to go to completion (i.e. increase hours up to overnight) in comparison to amplifications that just went for a few hours. This ensures that all of the nucleotides in the reaction are incorporated into DNA. We recommend 150-350 ng of TempliPhi amplified DNA per sequencing reaction.
4. What is in the Sample Buffer, Reaction Buffer and Enzyme Mix in the TempliPhi 100/500 kits 25-6400-10 and 25-6400-50?
Sample Buffer is 10 mM Tris-HCl pH 8.2, 0.5 mM EDTA containing random hexamers. Reaction Buffer contains salts, dNTPs and buffer. Enzyme Mix is a proprietary buffer containing f29 DNA polymerase and random hexamers.
5. What is in the TempliPhi Premix and Denature Buffer in the TempliPhi DNA Sequencing Template Amplification Kit 25-6400-01?
TempliPhi Premix composition is proprietary. Denature Buffer is 10 mM Tris-HCl pH 8.2, 0.5 mM EDTA.
6. What is the stability of TempliPhi 100/500 kits 25-6400-10 and 25-6400-50?
Storage at – 70 ºC up to a period of 10 months from the date of manufacture is recommended. The kit can also be stored at – 20 ºC up to one month from date of manufacture.
7. What is the stability of TempliPhi DNA Sequencing Template Amplification Kit 25-6400-01?
The TempliPhi Premix is stable at –70 ºC for 6 months from the date of manufacture. The kit components must freeze solid during storage. Do not store the TempliPhi Premix at –20 ºC. Avoid repeated freeze thawing. The denature buffer and positive control DNA are stable at –70 ºC or –20 ºC for 12 months from the date of manufacture.
8. How can I refreeze TempliPhi DNA Sequencing Template Amplification Kit 25-6400-01?
Place the kit in – 70 ºC and make sure that the TempliPhi Premix and denature buffer freeze to solid form by visual confirmation.
9. Can TempliPhi amplification be carried out at room temperature?
Yes. There is very little difference in TempliPhi amplification between 30 ºC and 25 ºC.
10. What is the hands-on time for a 96-well plate?
Typically it takes 20 min to set up the 96-well plate reactions. If this process is automated, it may take less hands-on time.
11. Can I store a TempliPhi reaction working stock at 4 ºC?
No. Any mixed and unused portions must be discarded.
12. Can I perform less than 10 reactions from TempliPhi 100 or 500 Amplification Kit at a time and store the remaining TempliPhi working reaction mix at – 20 ºC or - 70 ºC?
Yes, as many or as few reactions as required may be performed but any mixed, unused portions of TempliPhi premix must be discarded and not stored.
13. Can I pick up less than one colony or uneven amounts from a colony for amplification?
Yes. Transferring a complete colony into a TempliPhi amplification reaction is not recommended. Use a robot pin or tooth pick to gently touch a colony and transfer into TempliPhi sample buffer. Alternatively transfer an entire colony in 10-100 µl of TE and transfer 1 µl of this into TempliPhi sample buffer.
14. Can I use colonies from an old plate to amplify DNA or glycerol stocks?
Yes. DNA from colonies on plates up to 14 days old (stored at 4 ºC) have successfully been amplified using TempliPhi kit. However, fresh colonies are recommended whenever possible. For glycerol stocks, dilute 1 µl of glycerol stock in 50 µl of TE or water ad use 0.5-1 µl of diluted stock per amplification reaction.
15. How much overnight grown culture is required for amplification?
Addition of 0.25-1 µl of overnight grown culture is sufficient for DNA amplification. We recommend using no more than 1 µl. Customers can use 1-2 µl of 1:10 diluted culture for successful amplification. In house studies indicate that as low as 0.03 µl of overnight grown culture was sufficient template for amplification.
16. Does the starting material effect amplification kinectics?
Yes. Amplification kinetics are slightly quicker using colonies than culture as used culture contains components inhibitory to Phi29 DNA polymerase. Equivalent amplified DNA quantity and quality is obtained from cultures and colonies if the recommended protocols are followed.
17. How much time does it take to complete the amplification reaction?
Amplification reactions of typical sized M13 or plasmids (up to 12 kb) are completed in 4-6 h with the TempliPhi 100 or 500 Amplification Kits (25-6400-10 & 25-6400-50) or 16-18 h with the TempliPhi DNA Sequencing Template Amplification Kit (25-6400-01). Amplification of large templates (fosmids, BACs) is completed in 16-18 h with TempliPhi 100 or 500 Amplification Kits.
18. Do I have to precipitate DNA after amplification?
No. TempliPhi amplified DNA can be transferred directly into sequencing reactions for DYEnamic ET Terminator or Applied Biosystem's BigDye terminator sequencing kits. A precipitation step may be helpful in cases where sequencing a template which requires more than 500 ng DNA. It is recommended that no more than 4 µl of unpurified TempliPhi amplified DNA is added to a 20 µl sequencing reaction.
19. Can I amplify GC rich DNA? AT rich DNA?
Yes. We have not seen any difference in amplification success rate or efficiency of amplification among various DNA templates.
20. Why do I get amplification in a negative control DNA tube?
TempliPhi reactions are extremely sensitive to any traces of DNA. Primers present in the TempliPhi reagents and/or any minute amounts of contaminating DNA from other sources can amplify and be detected.
21. Can I use amplified DNA for sequencing using BigDye chemistry and ABI instruments?
Yes. Excellent results have been obtained using Applied Biosystem's BigDye terminator sequencing kits and sequencing instruments.
22. Does TempliPhi work with BACs?
Yes. We recommend using TempliPhi 100 or 500 Amplification Kits (25-6400-10 & 25-6400-50) for amplifying BACs and other large templates. A modified amplification protocol for large constructs can be found in the protocol booklet that accompanies these kits. During heat lysis, BACs are not released as efficiently as plasmids; amplification directly from BAC colonies results in higher background amplification. Included with the TempliPhi 100 and 500 Amplification Kits is a protocol for rapid miniprep purification of BAC templates that allows consistent and robust amplification. Please see our website for a separate Application note on the amplification of BACs with TempliPhi DNA Amplification Kits.
23. Can I clone the TempliPhi DNA products?
Yes, DNA amplified from TempliPhi reactions can be used for cloning after simple processing. DNA can be digested with single cutter restriction enzyme, ligated and then used for transformations. Phi29 DNA polymerase is a proofreading enzyme and the product of amplification is mostly double stranded.
24. What is the fidelity of Phi29 DNA polymerase?
Phi29 DNA polymerase has an error rate of 1 in 106-107 nucleotides3.
25. Can I amplify linear DNA with the TempliPhi DNA Sequencing Template Amplification Kit?
TempliPhi DNA Amplification Kits were developed specifically to prepare circular templates for DNA sequencing. The GenomiPhi DNA Amplification kit should be used for amplifying linear templates.
26. Can I obtain Phi29 DNA polymerase without hexamers?
No. Phi29 DNA polymerase is not available as a stand alone product.
27. Can I quantitate TempliPhi amplified DNA by UV absorbance?
No. Unused hexamers and nucleotides in the reaction will contribute to the absorbance measurement. Quantitate the amplified DNA using a double-stranded quantitation reagent such as PicoGreen (Molecular Probes). | 
