FastDigest PfeI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Restriction enzymes BseRI (New England Biolabs, Ipswich, MA), Cfr42I, and Fast Digest TfiI (PfeI) restriction enzyme (Fermentas/Thermo Fisher Scientific, Vilnius, Lithuania) was used for RFLP digestion for −216G>T, −191C>A, and 181946C>T, respectively (Table 1).	Products of restriction were detected by electrophoresis: for −191C>A and 181946C>T on 3 % agarose gel and for −216G>T on 8 % polyacrylamide gel.

Protocol tips
Restriction enzymes BseRI (New England Biolabs, Ipswich, MA), Cfr42I, and Fast Digest TfiI (PfeI) restriction enzyme (Fermentas/Thermo Fisher Scientific, Vilnius, Lithuania) was used for RFLP digestion for −216G>T, −191C>A, and 181946C>T, respectively (Table 1).
Downstream tips
Products of restriction were detected by electrophoresis: for −191C>A and 181946C>T on 3 % agarose gel and for −216G>T on 8 % polyacrylamide gel.

Publication protocol

"EGFR polymorphisms −216G>T and −191C>A were genotyped using the PCR-RFLP method according to Liu et al.
[22], with optimisation strategy and final conditions already described in Obradovic et al. [23]. Briefly, the temperature profile of PCR using KAPATaq HotStart PCR Kits
(Kapabiosystems, Boston, MA, USA) was initial denaturation at 95 °C for 5 min, cycling steps (×45) of denaturation
at 94 °C for 30 s, annealing at 63 °C for 30 s, extension at
72 °C for 60 s, than final extension at 72 °C for 7 min. The
total volume of PCR reaction was 25 μl, with 1 μl genomic
DNA, 0.4 μl each primer, 0.2 mM each dNTPs, 5 %
DMSO, and 1 U KAPA Taq DNA polymerase in 1× PCR
buffer A (with 1.5 mM MgCl2). Detection of 197 bp PCR
products was performed by gel electrophoresis with
ethidium bromide stained on 2 % agarose gel.
181946C>T (rs2293347) was genotyped according to Ma
et al. [20], with modifications. Namely, temperature profile
of PCR using KAPA Taq HotStart PCR Kits was initial
denaturation at 95 °C, 5 min; 45 cycles of denaturation at
94 °C, for 30 s; annealing at 55 °C, for 30 s; extension at
72 °C, for 60 s; and final extension at 72 °C, for 7 min. PCR
was performed in total volume of 25 μl, with 1 μl genomic
DNA, 0.4 μМ of each primer, 0.2 mМ each dNTPs, magnesium concentration was adjusted for 1.7 mМ MgCl2, and
1 U KAPA Taq DNA polymerase in 1× PCR buffer A. A
detection of 244-bp PCR products was performed by gel
electrophoresis with ethidium bromide stained on 2 % agarose gel.
Restriction enzymes BseRI (New England Biolabs,
Ipswich, MA), Cfr42I, and Fast Digest TfiI (PfeI) restriction
enzyme (Fermentas/Thermo Fisher Scientific, Vilnius,
Lithuania) were used for RFLP digestion for −216G>T,
−191C>A, and 181946C>T, respectively (Table 1). Products
of restriction were detected by electrophoresis: for −191C>A
and 181946C>T on 3 % agarose gel and for −216G>T on 8 %
polyacrylamide gel."

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