Efficient isolation of high quality nucleic acids from different tissues of Taxus baccata L.
Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and beta-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A(260)/A(280) and A(260)/A(230) ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100-300 microg/g leaf and stem tissue and total RNA with an average yield of 20-30 microg/g cell culture and 80-100 microg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.
Abbasi Kejani
Abolghasem
A
Agricultural Biotechnology Research Institute-Central Region of Iran (ABRICI), Isfahan, Iran.
Hosseini Tafreshi
Sayed Ali
SA
Khayyam Nekouei
Sayed Mojtaba
SM
Mofid
Mohammad Reza
MR
D002458:Cell Fractionation / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D018744:DNA, Plant / Q000302:isolation & purification; D004526:Efficiency; D009696:Nucleic Acids / Q000302:isolation & purification*; D011786:Quality Control; D018749:RNA, Plant / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020946:Taxus / Q000737:chemistry* / Q000235:genetics*
D018744:DNA, Plant; D009696:Nucleic Acids; D018749:RNA, Plant