Six commonly used restriction endonucleases (REs) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The res...Six commonly used restriction endonucleases (REs) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement for addi- tional magnesium supplemented as activator, REs, except EcoR I appeared star activity, completely digested unmethylated lambda DNA af- ter overnight incubation in PCR buffer and functioned as equally well as in recommended Restriction Enzyme Buffer provided with each enzyme; all REs tested completely digested PCR products in PCR buffer, it implied digestion of PCR products may often be performed di- rectly in the PCR tube without the requirement for any precipitation or purification steps; and the concentration of MgCl2 from 2.5 mmol·L-1 to 10 mmol·L-1 did not significantly affect activity of REs in PCR buffer. This simplified method for RE digestion of PCR prod- ucts could have applications in restriction fragment length polymorphism (RFLP) analysis and single-stranded conformational polymor- phism (SSCP) analysis of large PCR products. However, usage of this procedure for cloning applications needs further data.展开更多
Contamination problems on DNA isolation from 'recalcitrant plant taxa' which is rich in polysaccharides have been commonly encountered in a wide range of research fields such as plant population biology, biodi...Contamination problems on DNA isolation from 'recalcitrant plant taxa' which is rich in polysaccharides have been commonly encountered in a wide range of research fields such as plant population biology, biodiversity, and molecular marker-assisted breeding. Here we present an improved protocol to extract DNA efficiently from dry or fresh leaves of a 'recalcitrant plant taxa', Betula alnoides Buch. Ham. ex D. Don in which three key steps are involved: 1) washing out most of polysaccharides and other secondary compounds with CTAB-free buffer from homogenate; 2) adoption of 3% CTAB rather than 2% CTAB in the exaction medium; and 3) using of high concentration of salt prior to DNA precipitation with isopropanol to remove residual polysaccharides. The isolated DNA has been proved suitable for RAPD-PCR amplification and restriction digestion. This modified procedure is simple, inexpensive and reliable, and is also applicable to many other plant taxa with high polysaccharides.展开更多
Somatic cells were prepared from sea snail enzyme digests of Porphyra yezoensis thalli. Us ing SDS - Proteinase K as extraction solution, total DNA was isolated from the somatic cells. The crude extracts of total DNA ...Somatic cells were prepared from sea snail enzyme digests of Porphyra yezoensis thalli. Us ing SDS - Proteinase K as extraction solution, total DNA was isolated from the somatic cells. The crude extracts of total DNA were purified with glassmilk, and the resulting DNA was of sufficient quality for digestion of restriction endonuclease. DNA bands were clearly observed in the restriction patterns of EcoRI, PstI and HaeIII respectively. The presence of DNA hands in the restriction pattern of total DNA indicated that the genome of Porphyra yezoensis may be small. Unexpectedly, using guanidinium isoth iocyanate and sarcosyl as extraction solution, a plasmid-like DNA band (2.3 Kb) was directly found in the isolated total DNA of Porphyra yezoensis. A very simple and convenient method for plasmid-like DNA isolation has been established.展开更多
基金This research was supported by the Fund for ICP Cup of the Northeast Forestry University and partially by the Key Project (NO. 96-20) of State Forestry Administration.
文摘Six commonly used restriction endonucleases (REs) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement for addi- tional magnesium supplemented as activator, REs, except EcoR I appeared star activity, completely digested unmethylated lambda DNA af- ter overnight incubation in PCR buffer and functioned as equally well as in recommended Restriction Enzyme Buffer provided with each enzyme; all REs tested completely digested PCR products in PCR buffer, it implied digestion of PCR products may often be performed di- rectly in the PCR tube without the requirement for any precipitation or purification steps; and the concentration of MgCl2 from 2.5 mmol·L-1 to 10 mmol·L-1 did not significantly affect activity of REs in PCR buffer. This simplified method for RE digestion of PCR prod- ucts could have applications in restriction fragment length polymorphism (RFLP) analysis and single-stranded conformational polymor- phism (SSCP) analysis of large PCR products. However, usage of this procedure for cloning applications needs further data.
文摘Contamination problems on DNA isolation from 'recalcitrant plant taxa' which is rich in polysaccharides have been commonly encountered in a wide range of research fields such as plant population biology, biodiversity, and molecular marker-assisted breeding. Here we present an improved protocol to extract DNA efficiently from dry or fresh leaves of a 'recalcitrant plant taxa', Betula alnoides Buch. Ham. ex D. Don in which three key steps are involved: 1) washing out most of polysaccharides and other secondary compounds with CTAB-free buffer from homogenate; 2) adoption of 3% CTAB rather than 2% CTAB in the exaction medium; and 3) using of high concentration of salt prior to DNA precipitation with isopropanol to remove residual polysaccharides. The isolated DNA has been proved suitable for RAPD-PCR amplification and restriction digestion. This modified procedure is simple, inexpensive and reliable, and is also applicable to many other plant taxa with high polysaccharides.
文摘Somatic cells were prepared from sea snail enzyme digests of Porphyra yezoensis thalli. Us ing SDS - Proteinase K as extraction solution, total DNA was isolated from the somatic cells. The crude extracts of total DNA were purified with glassmilk, and the resulting DNA was of sufficient quality for digestion of restriction endonuclease. DNA bands were clearly observed in the restriction patterns of EcoRI, PstI and HaeIII respectively. The presence of DNA hands in the restriction pattern of total DNA indicated that the genome of Porphyra yezoensis may be small. Unexpectedly, using guanidinium isoth iocyanate and sarcosyl as extraction solution, a plasmid-like DNA band (2.3 Kb) was directly found in the isolated total DNA of Porphyra yezoensis. A very simple and convenient method for plasmid-like DNA isolation has been established.
