Recovery of bacterial DNA from soil with growth of wild mushroom

Chan, Weng Hoy (2005) Recovery of bacterial DNA from soil with growth of wild mushroom. Universiti Malaysia Sabah. (Unpublished)

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Abstract

Bacterial DNA was isolated from two types of soil sample near the growth of wild mushroom found in Universiti Malaysia Sabah campus area and were studied by using 16S ribosomal DNA (16S rDNA) assay. Two mushroom species. the Calvalia gigantean and another unknown mushroom species were discovered. The soil samples were undergone direct DNA extraction to recover the total DNA using a modified protocol from Zhou el al., (1996). The extracted DNA was brownish in colour due to the co-extraction of humic substances. The extracted DNA was then subjected to gel electrophoresis to check for the quality of the extracted DNA while compare to the marker, Lambda HindIII. From the gel electrophoresis. the extracted DNA was in expected size which was bigger than 23kb. After the DNA extraction, the DNA was subjected to purification seen the present of humic substances which was the inhibitor for the polymerase chain reaction (PCR) amplification in the DNA samples. The DNA samples were purified by using DNA Grade HTP Hydroxyapatite minicolumn method which was a modified protocol from Purdy el a/., (1996). From the result showed in the gel electrophoresis, the concentration of first elution DNA was higher compare to second elution. Between the two DNA samples from two different types of soil sample, the first mushroom's soil purified DNA was in white colour while the second mushroom's soil purified DNA was light brown in colour. This showed that there were still humic substances contained in the second mushroom's soil purified DNA. Both purified DNA were then subjected to PCR amplification by using 16S rDNA universal primer to amplify the conserve region in the genome of microorganisms. The DNA samples were diluted in 10X, 100X and 500X to overcome the problem of inhibition causing by the humic substances. From the PCR result obtained, only the first mushroom's soil DNA sample at 10X dilution showed the amplification and the result was reproducible. While for other DNA samples, there was no amplification showed. This was mainly due to the inhibition cannot be overcome by doing dilution for second mushroom's soil sample. As the conclusion, DNA should be pure enough and in good condition in order to give a high quality of PCR product.

Item Type: Academic Exercise
Uncontrolled Keywords: bacterial DNA, soil, wild mushroom, modified protocol, extraction, PCR amplification
Subjects: Q Science > QK Botany
Q Science > QR Microbiology > QR75-99.5 Bacteria
Divisions: SCHOOL > School of Science and Technology
Depositing User: Unnamed user with email storage.bpmlib@ums.edu.my
Date Deposited: 17 Mar 2015 06:49
Last Modified: 24 Oct 2017 06:44
URI: http://eprints.ums.edu.my/id/eprint/10589

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