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FAQs
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VeriFi™ Library Amplification Mix FAQs
Can I do multiplex PCR with VeriFi™ Library Amplification Mix?
Yes. For most multiplex experiments we recommend using VeriFi Hot Start Polymerase & Mixes (PB10.45-PB10.47 product catalogue numbers). However, VeriFi™ Library Amplification Mix is also well suited for this purpose, particularly when one or more of the multiplexed targets is GC-rich or contains difficult or repetitive sequence regions.
Can I use VeriFi™ Library Amplification Mix for methylation studies/amplification of bisulfite-treated DNA?
No. Pfu polymerases can only amplify uracil-containing targets after mutagenesis, which compromises PCR fidelity.
Can I use VeriFi™ Library Amplification Mix for standard PCR rather than library amplification?
Yes, please follow the relevant instructions in the product manual. VeriFi™ Library Amplification Mix can be used in standard 3-step or 2-step cycling programs and is particularly well suited to amplifying targets with very high or very low GC content.
Can VeriFi™ Library Amplification Mix be used in single cell NGS experiments?
VeriFi™ Library Amplification Mix can be used for PCR-based enrichment of any type of DNA sequencing library.
Do I need to include a purification step between adaptor ligation and library amplification with VeriFi™ Library Amplification Mix?
We recommend including a purification step after adaptor ligation and size selection. This eliminates any potential buffer incompatibility and ensures that un-ligated adaptors are not amplified during library PCR enrichment.
I am struggling to amplify a target with high GC content, what should I do?
Increase denaturation temperature to 98-100 °C and increase the extension time.
My amplified library is of poor quality, how can I improve it?
Increase denaturation temperature. Optimise annealing temperature and extension time. Optimise the amount of primers and template in the reaction. Make sure that the starting material is high quality.
What is the maximum target length I can amplify with VeriFi™ Library Amplification Mix?
We have successfully amplified a 12 kb fragment containing GC-rich regions. Longer targets may also be successfully attempted.
What is the minimum template amount I can use for successful library amplification with VeriFi™ Library Amplification Mix?
Do not use less than 10 ng template for NGS library amplification. A key factor in acquiring a high-quality amplified library is limiting the number of PCR cycles used. Using a higher number of cycles leads to greater chances of PCR induced errors and bias.
What NGS sequencing platforms can I use VeriFi™ Library Amplification Mix for?
VeriFi™ Library Amplification Mix has been extensively tested and validated on Illumina® sequencing platforms with the P5 and P7 Illumina® primers. The mix should work equally well in amplifying libraries with different adaptor sequences, intended for use on other sequencing platforms (e.g., Pac Bio and Oxford Nanopore Technologies), but this needs to be tested.
