I am grateful to J. Perrault (p.c.) for pointing out to me why my RT-PCR did not work when I
followed the more-standard advice to heat-denature my RNA template. I turns out it is just not necessary,
and I have amplified several cDNA targets up to 12 kb by skipping that step (Barnes & Ivanova,
unpublished).
Conflict of interest notice: The author is a for-profit/loss developer and provider of
Klentaq1 and improvements to PCR technology.
References
Barnes, W.M. (1994) PCR amplification of up to 35 kb DNA with high fidelity and high
yield from l bacteriophage templates, Proc. Natl. Acad. Sci. 91, 2216-2220.
Barnes, W.M. (1994b) Tips and tricks for long and accurate PCR, TIBS 19:342.
Barnes, W.M. (1995) U.S. Patent No. 5,436,149, Thermostable DNA polymerase with
enhanced thermostability and enhanced length and efficiency of primer extension.
Baskaran, N., Kandpal, R.P., Bhargava, A.K., Glynn, M.W., Bale, A., & Weissman, S.M.
(1996) Uniform amplification of a mixture of deoxyyribonucleic acids with varying GC
content, Genome Research 6:633-638.
Hogrefe HH. Hansen CJ. Scott BR. Nielson KB (2002) Archaeal dUTPase enhances PCR
amplifications with archaeal DNA polymerases by preventing dUTP incorporation. Proc.
Natl. Acad. Sci. 99: 596-601.
Lasken RS. Schuster DM. Rashtchian A. (1996) Archaebacterial DNA polymerases
tightly bind uracil-containing DNA. Journal of Biological Chemistry 271:17692-17696.
上一篇：Standard LA PCR Protocol 下一篇：LA PCR Protocol (Revised from Takara Shuzo Co., Ltd.)
共6页: 上一页 [1] [2] [3] [4] [5] 6 下一页
