Optimization of DNA isolation and peR parameters in Myristica sp. and related gen era for RAPD and ISSR analysis.
DSpice at Indian Institute of Spices Research
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Creator |
SHEEJA, T E
JOHNSON, GEORGE K JESSY JEROME SANDEEP VARMA, R SYAMKUMAR, S KRISHNAMOORTHY, B PARTHASARATHY, V A |
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Date |
2010-08-09T06:28:50Z
2010-08-09T06:28:50Z 2008 |
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Identifier |
Journal of Spices and Aromatic Crops Volume 17 (2) : 91-97 (2008)
http://hdl.handle.net/123456789/532 |
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Description |
An efficient protocol for isolation of DNA from wild and related genera of Myristica rich in polysaccharides and polyphenols was developed. The protocol utilizes CTAB (3%), 1.5% PVP and 0.3% B-mercaptoethanol for isolation and RNase and phenol chloroform extraction for purification. The yield of DNA ranged from 25-175 flg/g of fresh leaf tissue, with Knema andamanica giving the highest yield. The present method yielded 10 times higher than the old methods. Characteristic patterns were generated on digestion of DNA by Ecod and Hind III restriction enzymes. PCR parameters were optimized using random primers (OPERON Technology, USA). DNA concentration at 20 ng/reaction, annealing temperature of 45°C, 0.3 mM dNTP in presence of 0.5 U of Taq DNA polymerase, and 2.0 mM MgCl and MJ Research z Gene Thermocycler was best. Successful amplification by ISSR and RAPD primers indicated that DNA is of good quality and free of polysaccharides and polyphenols.
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Format |
491594 bytes
application/pdf |
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Language |
en
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Publisher |
Indian Society for Spices
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Subject |
DNA isolation
optimization PCR polymorphism restriction digestion |
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Title |
Optimization of DNA isolation and peR parameters in Myristica sp. and related gen era for RAPD and ISSR analysis.
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Type |
Article
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