(0.5pg/uL, TIB MOLBIOL, Germany) and template DNA were added (2 uL each).
The quality and quantity of extracted DNA was tested by comparing the sample with known standards of lambda DNA
on 1% agarose gel electrophoresis.
EcoRI/Hind-III double digest was used as a molecular size marker.
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
We have found that a one second denaturation at 92 [degrees]C is sufficient for a variety of PCR products amplified with iQ supermix, including the 83.5% 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).
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.
A lane of HindIII-digested lambda DNA
was also loaded onto each gel to provide molecular weight markers.
The cut fragments were loaded into an electrophoresis gel and measured, analyzed, and compared to Lambda DNA
. I hypothesized that the lead dioxide present in the nutrient agar would affect the DNA fragment lengths of E.