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Leaf storage conditions
and genomic DNA isolation efficiency in Ocimum
gratissimum L. from Kenya
Lexa G. Matasyoh1*,
Francis N. Wachira2, Miriam G. Kinyua3,
Anne W. Thairu Muigai1 and Titus K. Mukiama4
1Department
of Botany, Jomo Kenyatta University of Agriculture and
Technology, P. O. Box 62000, Nairobi, Kenya.
2Department
of Biochemistry, Egerton University, P. O. Box 536, Njoro,
Nakuru, Rift Valley, Kenya.
3Kenya
Agricultural Research Institute, P.O. Njoro, Nakuru, Kenya.
4Department
of Botany, Nairobi University, P. O. Box 29053, Nairobi,
Kenya.
*Corresponding author. E-mail:
lexa111@hotmail.com.
Tel: +254 722614696.
Accepted
16 January, 2008 |
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Storage of plant tissues for DNA is important to avoid
degradation of DNA. Preliminary studies were conducted on
Ocimum gratissimum L. in order to establish the storage
conditions for the collected samples before DNA extraction.
Secondly, the aim was to determine the best protocol for the
extraction of high quality DNA, which would later be used
for molecular analysis. DNA was extracted from the samples
one month after field sampling. During the DNA extraction,
four protocols were used; the modified hexadecyltrimethyl
ammonium bromide (CTAB) mini preparation method described by
Doyle and Doyle (1990), with reductants either
mercaptoethanol or dithiothreitol; the modified sodium
dodecyl sulphate (SDS) mini preparation method of
Edwards et al. (1991) with
redundant either mercaptoethanol or dithiothreitol. The DNA
was purified, treated with RNase, quantified and examined
for intactness using gel electrophoresis method. Good
quality and high yield DNA could only be extracted with the
buffer containing the detergent SDS and the reducing agent
dithiothreiotol.
Key
words:
Ocimum gratissimum L., Sodium dodecyl sulphate (SDS),
hexadecyltrimethl ammonium bromide (CTAB), dithiothreitol,
mercaptoethanol. |
