Best Polymerase for Long Range PCR followed by Nextera XT kit

[jesstilla]

Hello, I am new to using Next Generation Sequencing.

I am hoping to do long range PCR (20-30 kb), followed by library prep with a Nextera XT kit, followed by sequencing with a MiSeq.

My PI has asked me to find a PCR enzyme that is appropriate for long PCR and will yield products that are appropriate for the next steps of my project. I'm not sure what criteria to look for in a good enzyme for this situation. Is there anything I should be on the look out for? In particular, he told me to look at the three enzymes offered by Life Technologies on this page http://www.lifetechnologies.com/us/e...gment-pcr.html and determine which one was the best for this purpose. I have already called the company, and they were not terribly helpful.

If anyone has suggestions, I would be very happy to hear them. If you could put them in as simple/basic language as possible, I would appreciate that, because I am new to this field.

Thanks so much!

[JackieBadger]

http://www.qiagen.com/products/catalog/assay-technologies/end-point-pcr-and-rt-pcr-reagents/qiagen-longrange-pcr-kit

[ATϟGC]

I just started long-PCR myself and I decided to try longamp hot start from NEB [https://www.neb.com/products/m0534-l...na-polymerase]. I use it at half the concentration reccomended in 25ul reactions, which ends up costing about $0.25 per reaction (~$125 for 500 units). My first reactions worked well for 12-20 Kb regions without any optimization.The product guide says it will amplify up to 30 Kb of lambda or human genomic DNA. I have no experience with other long range PCR polymerases.

[Simone78]

why not using KAPA HiFi HotStart ready Mix. it comes in a ready-to-use 2X mix and it´s a very robust enzyme, tolerating a range of annealing T and salt concentration. It has been shown to be able to amplify GC regions, where other Pol are failing. We used it all the time to amplify full-length cDNA libraries from single cells.

[megsmcbride]

I have been using Takara LA Taq, which worked well to amplify products btw 10-18kb for the first few times, but I am finding reproducibility is low in long range pcrs. Or at least that is what I am experiencing. Has anyone else experienced this? I am going to try the LongAMP HS Taq..and see if that is better. One thing I did notice is that using a two-step PCR protocol worked much better than 3-step pcr.