PCR reaction and DNA digestions. Ce

PCR reaction and DNA digestions. Cells were directly collected
from a fresh yeast colony using a yellow tip and
suspended in 100 pl PCR reaction mix containing 0.5 pM
primer ITS 1 (5' TCCGTAGGTGAACCTGCGG 3'),
0.5 pM primer ITS4 (5' TCCTCCGCTTATTGATATGC
3'), 10 pM deoxynucleotides, 1.5 mM MgCl, and 1 x buffer
(MAD-GEN). The suspension was heated at 95 "C for
15 min in a Progene (Techne) thermocycler. One unit of
DNA Polymerase SuperTherm (MAD-GEN) was then
added to each tube. PCR conditions were as follows : initial
denaturation at 95 "C for 5 min; 35 cycles of denaturing at
94 "C for 1 min, annealing at 55.5 "C for 2 min and extension
at 72 "C for 2 min; and a final extension at 72 "C for 10 min.
PCR products (10 p1 or approximately 0.5-1.0 pg) were
digested without further purification with the restriction
endonucleases CfoI, HaeIII and Hinff (Boehringer
Mannheim), although endonucleases AM, DdeI, ScrFI or
Tag1 were additionally used in some particular cases. The
PCR products and their restriction fragments were separated
on 1-4 YO and 3 YO agarose gels, respectively, with 1 x TAE
buffer. After electrophoresis, gels were stained with ethidium
bromide, visualized under UV light and photographed
(Image Master, Pharmacia). Sizes were estimated by comparison
against a DNA length standard (100 bp ladder,
Gibco-BRL).

ผลลัพธ์ (อังกฤษ) 2:

PCR reaction and DNA digestions. Cells were directly Collected
from a Fresh yeast Colony using a Yellow Tip and
suspended in 100 PL PCR reaction Mix containing 0.5 pM
Primer ITS 1 (5 'TCCGTAGGTGAACCTGCGG 3'),
0.5 pM Primer ITS4 (5 'TCCTCCGCTTATTGATATGC
3 '), 10 pM Deoxynucleotides. , 1.5 mM MgCl, and 1 x buffer
(MAD-GEN). The suspension was heated at 95 "C for
15 min in a Progene (Techne) Thermocycler. One UNIT of
DNA Polymerase SuperTherm (MAD-GEN) was then
added to each tube. PCR conditions were As follows: initial
denaturation at 95 "C for. 5 min; 35 Cycles of denaturing at
94 "C for 1 min, annealing at 55.5" C for 2 min and Extension
at 72 "C for 2 min; and a Final Extension at 72" C for 10 min.
PCR products (10 P1 or approximately 0.5. -1.0 PG) were
digested without further Purification with the Restriction
endonucleases CfoI, and Hinff HaeIII (Boehringer
Mannheim), endonucleases Although AM, DdeI, ScrFI or
tag1 were additionally used in Some Cases particular. The
PCR products and their Restriction fragments were Separated
on 1-4 YO YO agarose gels and 3, respectively, with 1 x TAE
buffer. After electrophoresis, gels were Stained with ethidium
bromide, visualized and photographed under UV Light
(Image Master, Pharmacia). Sizes were estimated by comparison
against a standard DNA Length (BP Ladder 100,
Gibco-BRL).

ผลลัพธ์ (อังกฤษ) 3:

PCR reaction and DNA digestions. Cells were directly collected
from a fresh yeast colony using a yellow tip and
suspended. In 100 pl PCR reaction mix containing 0.5 pM
primer ITS 1 (5 'TCCGTAGGTGAACCTGCGG 3'),
0.5 pM primer ITS4 (5 'TCCTCCGCTTATTGATATGC
3'), 10 pM. Deoxynucleotides 1.5 MgCl and, mM, 1 x buffer
(MAD-GEN). The suspension was heated at 95 "C for
.15 min in a Progene (Techne) thermocycler. One unit of
DNA Polymerase SuperTherm (MAD-GEN) was then
added to each, tube. PCR conditions were as follows: initial
denaturation at 95 "C for 5 min; 35 cycles of denaturing at
94" C for, 1 min annealing. At 55.5 "C for 2 min and extension
at 72" C for 2 min; and a final extension at 72 "C for 10 min.
PCR products (10 P1 or. Approximately 0.5-1.0 PG) were
digested without further purification with the restriction
endonucleases CfoI HaeIII and, Hinff (Boehringer
Mannheim),. Although endonucleases AM DdeI ScrFI,, or
Tag1 were additionally used in some particular cases. The
PCR products and their. Restriction fragments were separated
on 1-4 YO and 3 YO, agarose gels respectively with 1, X TAE
buffer, After electrophoresis.Gels were stained with ethidium
bromide visualized under, UV light and photographed
(Image, Master Pharmacia). Sizes were. Estimated by comparison
against a DNA length standard (100, BP ladder
Gibco-BRL).