PCR Enrichment - Replace TruSeq PPC and PMM?!

[Akira]

Hi all, NGS newbie here. I know this has been discussed in other threads, but I don't really understand. I have already had the sample until ligation part (using TruSeq DNA Sample Prep Kit), I would like to amplify it using my own primers, instead of using TruSeq PCR Primer Cocktail (PPC). But I am not sure what is the optimized concentration of the primers used and what is the content of TruSeq PPC. Say if I prepare my own primer mix, maybe 25uM primers, can I use them together with the TruSeq Primer Master Mix? Anyone tried this before? Or can I perform normal PCR reaction using normal PCR reagents? What is the polymerase used in TruSeq Primer Master Mix? If I were to buy others to replace TruSeq PMM, what is the best kit? Aww I have too many questions in my mind now, anyone can help?

[pbluescript]

I have had good results with PCR master mixes from both NEB and Kapa:

http://www.neb.com/nebecomm/products/productM0531.asp

http://www.kapabiosystems.com/products/name/kapa-hifi-pcr-kits

When you say your own primers, do you mean your own specific sequence or using the same sequence as Illumina, but just getting them from another source? I've never tried the former, but the latter works well.

[Akira]

Many thanks for the info! How about the TruSeq PCR Master Mix? Try that with own custom made primer before? I have that with me now, and it takes time if I were to order from NEB or KAPA, so I am wondering if I can use that. But well, I don't want to waste my samples if that TruSeq PMM does not work. I have common Promega Tag polymerase, dNTP, MgCl2 etc etc for routine normal PCR use, can I use that? Or it is recommended to use some high fidelity polymerases?

Normally for own custom made primers, how much is the concentration used in the PCR reaction?

By the way, my custom made primers are not the same as the Illumina one, I have own sequence to target.

Sorry if my questions sound stupid, I am still quite confused with this sample prep thing.

[pbluescript]

Stick with a high fidelity polymerase. No need to create more sequencing errors than you are already going to get.

If you are using your own primers, I would be sure to do some optimization before you use them on the libraries you are going to sequence. I've never done this though, so I'm not sure of the best way to proceed. Perhaps someone else could suggest something?