Long-range PCR

[ellenthomas]

I am trying to prepare multiple samples with Invitrogen's Sequalprep long range PCR kit for sequencing on a 454. Does anyone have any experience of this, for example how much do you need to quality control your PCR products (for specificity etc) before quantification and pooling?

[vasvale]

Hi

I don't know about this and I'd like to hear if you find out. Can you please tell me how you process the PCR products for subsequent sequencing or can you point out literature?

thanks

vasvale

[ellenthomas]

I'm using the Sequalprep LR-PCR kit and Normalisation plates, then using Parallel Tagged Sequencing (Meyer et al 2008, Nat Protocols 3 (2) 267-278)

[ATGC_man]

Hi Ellenthomas !

Nice ! have you already started your test ? I'd like to hear more about that, may i will go in this way...

[ATGC_man]

Hello

Which software will you use to design your primers for long PCR ?

[ellenthomas]

I just use Primer 3 which is free on the web. There are primer design instructions in the Sequalprep kit.

[rzhuo]

Hi there,
I'm also using the SequalPrep Kit and I do a simple sanger sequencing of the ends of my fragment using my LR PCR primers to verify my fragments.

[james hadfield]

We are using SeqalPrep for lr-PCR and it is working. However there are lots of questions that we are thinking about as the read coverage is nowhere near smooth across the region and although I expected some varialbility I did not think it would be as high as it is.
JAmes.

[The_Roads]

We're doing something different but also have a lot of problem with uneven non-random coverage. If anyone knows of any tweeks to smooth coverage I'd be interested to hear about them.

[nighteffect]

Bad efficiency

We've tried using this kit for enrichment of regions around 10-12kb. The efficiency for this was near zero. We downscaled it down to 3-7kb and are starting to get amplification efficiency around 30-50%.
For our purposes this kit is waste of money, especially because of i) the price, and ii) nonexistent customer support. We wrote several times to speak to people who actually KNOW how this kit works, but we got general answers about how to design primers.....
Well, the best option for us would be raindance, but who can afford this...

[TonyBrooks]

Any advice on how to purify LR-PCR products before shearing?
Would Ampure beads work or maybe Qiaquick columns?

[The_Roads]

standard Sigma pcr cleanup columns and Clontech/Macherey-Nagel nucleospin columns have both worked fine for us to supply template to our core. i am pretty certain they do not do any further clean up prior to fragmentation.