User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/13

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Amplifying gene rich region of CCA3

  • Using AV as positive control for primers

PCR conditions

Reaction mixture conc. 1x 17
water 5.35 90.95
LA buffer 10x 1 17
dNTP mix 2.5 mM each 1.6 27.2
Primer mix 5 uM each 1
TakaraLA 5U/ul 0.05 0.85
template 1 ng/ul 1
8
  • Primer sets (first row of diluted plate):
Avhsp-1_1-1F Avhsp-1_4-1F Avhsp-1_7-1F Avhsp-3_3-1F Avhsp-4_3-1F Avhis-1_2-1F Avhis-2_2-1F Avhis-3_2-1F
  • 94C,1 min (98C,10 sec;65C 10 min)x30

Gel

Asm100413.jpg

Gene-rich region of CC A3 - the rest of the primers

Program

Reaction mixture conc. 1x 44
water 5.35 235.4
LA buffer 10x 1 44
dNTP mix 2.5 mM each 1.6 70.4
Primer mix 5 uM each 1
TakaraLA 5U/ul 0.05 2.2
template 1 ng/ul 1 44
8
  • primer orientation
' 1 2 3 4 5 6 7 8
a Avhsp-1_1-2F Avhsp-1_4-2F Avhsp-1_7-2F Avhsp-3_3-2F Avhsp-4_3-2F Avhis-1_2-2F Avhis-2_2-2F Avhis-3_2-2F
b Avhsp-1_2-1R Avhsp-1_5-1R Avhsp-3_1-1R Avhsp-4_1-1R Avhsp-4_4-1R Avhis-1_3-1R Avhis-2_3-1R Avhis-3_3-1R
c Avhsp-1_2-2F Avhsp-1_5-2F Avhsp-3_1-2F Avhsp-4_1-2F Avhsp-4_4-2F Avhis-1_3-2F Avhis-2_3-2F Avhis-3_3-2F
d Avhsp-1_3-1F Avhsp-1_6-1F Avhsp-3_2-1F Avhsp-4_2-1F Avhis-1_1-1F Avhis-2_1-1F Avhis-3_1-1F Avhsp-4_1-1F
e Avhsp-1_3-2F Avhsp-1_6-2F Avhsp-3_2-2F Avhsp-4_2-2F Avhis-1_1-2F Avhis-2_1-2F Avhis-3_1-2F Avhsp-4_1-2F
f Avhsp-4_2-1F Avhsp-4_2-2F
  • 94C,1 min (98C,10 sec;65C 10 min)x30

Gel

Asm100412b.bmp

Alien Genes New Primer Screen

Reaction mixture conc. 1x x16.5 ' lane '
Temp ?? 2 27 Ithaca 2 A3
TopTaq Buffer 10x 1 16.5 28 Ithaca 2 A4
dNTP mix 10 mM each 0.2 3.3 29 Ithaca 2 A5
Primer mix (HCO/LCO) 5 uM each 1 16.5 37 Tsukuba 3 D2
TopTaq 5 U/ul 0.05 0.825 40 Woods Hole 1 A6
Milli Q 5.75 94.875 43 Yokohama B5
10 ul 47 Harvard A1
48 Ithaca 2 A6
PCR cycle 50 Tsukuba 1 B5
94C 3 min 51 Yokohama A2
55 Yokohama B2
98C 10 sec 56 Yokohama A4
60C 3 min 61 Pennsylvania(PA) B3
x35 62 Ithaca1 C3
75 Baltimore A5
72C 5 min 76 Pennsylvania (PA) B1

0072.jpg