Long accurate PCR-help

[Paul N Hengen]

brett (brett at BORCIM.WUSTL.EDU) wrote:

| For some time I am trying to amplify large fragment from Streptomyces
| linear plasmid (16 kb). Apparently I wasn=92t very successful. Though I
| changed different parameters, denaturation temp., annealing temp.,
| enzyme concentration ( I am using Klentaq LA as well as Elongase from
| LTI), added betaine or DMSO, I still get nothing. Did anyone have luck
| with large G+C rich template. If someone has any useful suggestion I
| will be very grateful.

> There was a thread some time ago about using betaine in G/C-rich PCRs, and
> I happen to know Wayne Barnes uses it in his LA procedure. 

Yes. A review of that discussion will be published in the next TiBS column:

@article{Hengen1997Juntibs,
author = "P. N. Hengen",
title = "Methods and reagents - Optimizing multiplex and {LA-PCR}
with betaine",
journal = "Trends in Biochemical Sciences",
volume = "22",
number = "6",
pages = "xxx-xxx",
month = "jun",
year = "1997"}

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