Extracted DNA was further purified using the phenol-chloroform method and quantified by analyzing DNA on 1% agarose gel using diluted uncut lambda DNA
Aliquots (1 [micro]L) of purified PCR products were quantified by comparison with serial dilutions of uncut lambda DNA
(Promega) in 1% agarose gels (Fig.
1U corresponds to the amount of enzyme required to digest 1 [micro]g of lambda DNA
in 1 h at 37[degrees]C in 50 [micro]l of assay buffer.
Phage Lambda DNA
was used as molecular weight marker.
Restriction digestion of Lambda DNA
and DNA from African individuals: To check for the effects of PCR components on the activity of enzymes, the digestion pattern of pure Lambda DNA
(taken as positive control of digestion) was analyzed with respect to Lambda DNA
spiked with PCR amplicon in three different forms: (a) untreated PCR amplicon spiked with Lambda DNA
; (b) salt precipitated PCR amplicon spiked with Lambda DNA
; and (c) column precipitated PCR amplicon spiked with Lambda DNA
5% GC, 505 bp PCR product in Figures 1 and 2, as well as a 64% GC, 150 bp PCR product in lambda DNA
(data not shown).
Lane 2: Lambda DNA
digested with Bst EII used as molecular weight marker.
If more detailed studies document that lambda DNA
indeed has a short half-life in human specimen matrices, then its range of applications will be limited.
Quantifying was done using agarose minigel electrophoresis with lambda DNA
as the standard.
coli that was exposed to lead showed very few fragments compared to Lambda DNA
The difference between states is not due to variation in DNA sequence, but rather variation in which regulatory proteins are bound to the control regions of the lambda DNA
The quantity of DNA was also confirmed by comparing with Quantification Standards Phage Lambda DNA
(Fermentas) on 0.