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A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.
| Content Provider | Semantic Scholar |
|---|---|
| Author | Edwards, Kirstin Johnstone, Carol J. Thompson, Corey |
| Copyright Year | 1991 |
| Abstract | The polymerase chain reaction (PCR) has revolutionised the rapid analysis of mammalian genomic DNA (1). However, PCR is less useful in the analysis of plant DNA due to the difficulties in extracting nucleic acids from limited amounts of plant tissue. We have developed a method for the rapid extraction of small amounts of plant genomic DNA suitable for PCR analysis. The method is applicable to a variety of plant species and has the added advantage of not requiring any phenol or chloroform extraction. Thus it is possible to complete an extraction within 15 minutes without handling any hazardous organic solvents. Samples for PCR analysis (usually leaf tissue) are collected using the lid of a sterile Eppendorf tube to pinch out a disc of material into the tube. This ensures uniform sample size and also reduces the possibilities of contamination arising from handling the tissue. DNA is extracted as follows: The tissue is macerated (using disposable grinders from Bel-art Products: Scienceware, Pequannock, NJ, 07440 USA. catalog no 992) in the original Eppendorf tube at room temperature, without buffer, for 15 seconds. 400 yl of extraction buffer (200 mM Tris HC1 pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) is added and the sample vortexed for 5 seconds. This mixture can then be left at room temperature until all the samples have been extracted (> 1 hour). The extracts are centrifuged at 13,000 rpm for 1 minute and 300 y\ of the supernatant transferred to a fresh Eppendorf tube. This supernatant is mixed with 300 /il isopropanol and left at room temperature for 2 minutes. Following centrifugation at 13,000 rpm for 5 minutes, the pellet is vacuum dried and dissolved in 100 /xl 1XTE. This DNA is stable at 4°C for greater than one year. 2.5 yX of this sample is sufficient for a standard 50 y\ PCR (Figure 1). When older tissue is used this may be increased to 25 /xl without any deleterious effect on the PCR. Using this protocol we have found it possible to process hundreds of individual samples in a single working day. |
| Starting Page | 1349 |
| Ending Page | 1349 |
| Page Count | 1 |
| File Format | PDF HTM / HTML |
| DOI | 10.1093/nar/19.6.1349 |
| Alternate Webpage(s) | https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/nar/19/6/10.1093/nar/19.6.1349/2/19-6-1349.pdf?Expires=1492444030&Key-Pair-Id=APKAIUCZBIA4LVPAVW3Q&Signature=MA0nVA-lnjRs-kO7RzeIj-T4APXGb12K95YjF8--ExsdpCh6lJmt24jz1VS5M1tO4c0M7p-mPA0F7VXqlbSLHA4LNdAiO1NUIs0357S6EjSKrVcIZB8koDen9SY8YrQunZl52fsOmU4k1BFkxut8~4xuxXSA9BqjTCQyXTgnodzxVQNJtxJkVNQpCNkSKR6kzkjVGS3~7R1iNt1izMAapG0fs249X8~iH7V3~qJhGPu4NIR1lLNPiO05Ll2XxGi50CHKqsF5UsKyzqSiNY0g7J0dakK7Iz4BVecaRJHfFwhtI7FSie57Rupuc7xX6fag0cjjtN4MMYI651Bx-nMlCw__ |
| PubMed reference number | 2030957 |
| Alternate Webpage(s) | https://doi.org/10.1093/nar%2F19.6.1349 |
| Journal | Medline |
| Volume Number | 19 |
| Issue Number | 6 |
| Journal | Nucleic acids research |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
