MnlI NEB#R0163

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Ten microliters of PCR product were digested with 5 units of the MnlI restriction enzyme (NEB, Beverly, MA) following the manufacturer’s instructions in a final reaction volume of 30 µL.	The restriction fragments were separated by gel electrophoresis in 1X sodium borate buffer (Faster Better Media, Hunt Valley, MD) on 4.0% NuSieve GTG agarose gels containing ethidium bromide; the gel was photographed on a UV transilluminator. The gel patterns for each sample were scored according to MnlI fragment sizes (Table 1 and Fig 1A).

Protocol tips
Ten microliters of PCR product were digested with 5 units of the MnlI restriction enzyme (NEB, Beverly, MA) following the manufacturer’s instructions in a final reaction volume of 30 µL.
Downstream tips
The restriction fragments were separated by gel electrophoresis in 1X sodium borate buffer (Faster Better Media, Hunt Valley, MD) on 4.0% NuSieve GTG agarose gels containing ethidium bromide; the gel was photographed on a UV transilluminator. The gel patterns for each sample were scored according to MnlI fragment sizes (Table 1 and Fig 1A).

Publication protocol

"This reaction yielded a 332-bp product, spanning from
np16,106 to 16,437 within the human mtDNA D-loop
region. Ten microliters of PCR product were digested with
5 U of the MnlI restriction enzyme (NEB, Beverly, MA)
following the manufacturer’s instructions in a final reaction
volume of 30 ll. The restriction fragments were separated by
gel electrophoresis in 19 sodium borate buffer (Faster Better
Media, Hunt Valley, MD) on 4.0% NuSieve GTG agarose
gels containing ethidium bromide; the gel was photographed
on a UV transilluminator. The gel patterns for each sample
were scored according to MnlI fragment sizes (Table 1;
Fig. 1a). Samples apparently not conforming to any single
pattern (scored as ‘‘atypical’’), or displaying multiple phenotypes, were re-run through the entire PCR-RFLP process
and re-evaluated to assure complete enzymatic digestion and
PCR fidelity. Somatic MnlI site mutation was defined as the
difference in MnlI RFLP pattern between tumor tissue and
the corresponding, adjacent, non-tumor tissue"

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