User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/20

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Nuclear Gene HS Takara PCR optimization

' ' ' ' ' ' AMP Lane Template Primer
HS Takara RXN Mixture conc 1X 6.5 1 71 Instagene A1
Milli Q water 1.1 7.15 2 71 Instagene A2
Takara HS Buffer 10X 2 13 3 71 Instagene A3
dNTP 2.5uM 0.8 5.2 4 71 Instagene A4
Primer Mix 5uM 4 5 71 Instagene A5
HS Taq 5U 0.1 0.65 6 71 Instagene A6
Template Instagene 2 13 7 72 Instagene A1
39 6.0ul / RXN 8 72 Instagene A2
A PCR cycle 9 72 Instagene A3
95C 3 min 10 72 Instagene A4
95C 5 sec 11 72 Instagene A5
60C 1 min 12 72 Instagene A6
x35 13 AV Instagene A1
14 AV Instagene A2
*AV Positive Control 15 AV Instagene A3
16 AV Instagene A4
17 AV Instagene A5
18 AV Instagene A6

All amps failed - repeat with 2.0ul Primer concentration as control


HS Takara RXN Mixture conc 1X 6.5 ' ' AMP Lane Template Primer
Milli Q water 1.1 7.15 1 71 Instagene A1
Takara HS Buffer 10X 2 13 2 71 Instagene A2
dNTP 2.5uM 0.8 5.2 3 71 Instagene A3
Primer Mix 5uM 4 4 71 Instagene A4
HS Taq 5U 0.1 0.65 5 71 Instagene A5
Template Instagene 2 13 6 71 Instagene A6
39 6.0ul / RXN 7 72 Instagene A1
A PCR cycle 8 72 Instagene A2
95C 3 min 9 72 Instagene A3
95C 5 sec 10 72 Instagene A4
60C 1 min 11 72 Instagene A5
x35 12 72 Instagene A6
13 AV Instagene A1
Positive Control MiX 14 AV Instagene A2
HS Takara RXN Mixture conc 1X 6.5 15 AV Instagene A3
Milli Q water 3.1 20.15 16 AV Instagene A4
Takara HS Buffer 10X 2 13 17 AV Instagene A5
dNTP 2.5uM 0.8 5.2 18 AV Instagene A6
Primer Mix 5uM 2 19 AV Instagene A1
HS Taq 5U 0.1 0.65 20 AV Instagene A2
Template Instagene 2 13 21 AV Instagene A3
52 8.0ul / RXN 22 AV Instagene A4
A PCR cycle 23 AV Instagene A5
95C 3 min 24 AV Instagene A6
95C 5 sec
60C 1 min
x35
*AV Positive Control


load 2.oul of sample

20April10.jpg

Cycle Sequence HCO/LCO PCR Product

      • 07April10 Ethanol Cleaned PCR Product - added 20ul more water = total 30ul -> used this for CS
CS Reaction mixture conc. 1x 90 '
Big Dye Terminator 0.5 45
Primer (FWD or RVS) 5uM 0.4 36
DMSO 0.1 9
5X Seq Buffer 5X 0.4 36
Water 3.6 324
Template * ? 1
450 5.0ul / RXN
* Ethanol Cleaned PCR product
Cycle Seq Program
96C 10 sec
50C 5 sec
60C 4 min
x60
CS Plate Source Plate Source Plate Well EX ID CS Plate Source Plate Source Plate Well EX ID
A1 Baltimore A1 1 D6 Baltimore A3 44
B1 Baltimore B1 2 E6 Baltimore A6 45
C1 Baltimore C2 3 F6 E. Windsor D4 46
D1 Baltimore C3 4 G6 Harvard A1 47
E1 E. Windsor A1 5 H6 Ithaca 2 A6 48
F1 E. Windsor A3 6 A7 Pennsylvania (PA) D4 49
G1 E. Windsor B1 7 B7 Tsukuba 1 B5 50
H1 E. Windsor B2 8 C7 Yokohama A2 51
A2 E. Windsor C2 9 D7 Baltimore C1 52
B2 Harvard A2 10 E7 Ithaca 1 A4 53
C2 Harvard A3 11 F7 Pennsylvania (PA) B6 54
D2 Ithaca 1 A1 12 G7 Yokohama B2 55
E2 Ithaca 1 A2 13 H7 Yokohama A4 56
F2 Ithaca 1 A3 14 A8 Ithaca 1 C4 57
G2 Ithaca 1 A5 15 B8 Ithaca 1 D5 58
H2 Ithaca 1 A6 16 C8 Baltimore C5 59
A3 Ithaca 1 B1 17 D8 E. Windsor A2 60
B3 Ithaca 1 B3 18 E8 Pennsylvania (PA) B3 61
C3 Ithaca 1 B5 19 F8 Ithaca 1 C3 62
D3 Ithaca 1 C1 20 G8 Ithaca 1 C6 63
E3 Ithaca 1 D1 21 H8 Harvard A4 64
F3 Ithaca 1 D3 22 A9 Ithaca 1 B4 65
G3 Ithaca 1 D4 23 B9 Ithaca 1 C5 66
H3 Ithaca 1 D6 24 C9 Baltimore B2 67
A4 Ithaca 2 A1 25 D9 Baltimore B3 68
B4 Ithaca 2 A2 26 E9 Baltimore B6 69
C4 Ithaca 2 A3 27 F9 E. Windsor C3 70
D4 Ithaca 2 A4 28 G9 Pennsylvania (PA) D6 71
E4 Ithaca 2 A5 29 H9 Baltimore B5 72
F4 Pennsylvania (PA) B4 30 A10 Ithaca 1 C2 73
G4 Tsukuba 1 A4 31 B10 Pennsylvania (PA) D5 74
H4 Tsukuba 1 A6 32 C10 Woods Hole 1 B3 E
A5 Tsukuba 1 B2 33 D10 Woods Hole 1 B5 F
B5 Tsukuba 1 B4 34 E10 Woods Hole 1 C4 G
C5 Tsukuba 1 C1 35 F10 Woods Hole 2 A6 H
D5 Tsukuba 1 C6 36 G10 Cape Cod (CC) A3 CCA3
E5 Tsukuba 3 D2 37 H10 Boston A1 A
F5 Tsukuba 3 D3 38 A11 Boston A4 B
G5 Tsukuba 3 D4 39 B11 Cape Cod (CC) A1 C
H5 Woods Hole 1 A6 40 C11 Woods Hole 3 A5 D
A6 Woods Hole 1 C2 41 D11 Woods Hole 3 C2 I
B6 Woods Hole 1 C3 42 E11 Baltimore A5 75
C6 Yokohama B5 43 F11 Pennsylvania (PA) B1 76

Extraction confirmation by PCR

InstaGene Matrix

Reaction mixture conc. 1x x8.5 ' No. Primer mix template
Temp (InstaGene extraction) ?? 2 17 1 AV10045 CC A3
TopTaq Buffer 10x 1 8.5 2 AV10045 Pennsylvania (PA) D6 (71)
dNTP mix 10 mM each 0.2 1.7 3 AV10045 Baltimore B5 (72)
Primer mix 5 uM each 1 4 AV10045 Tsukuba3 D4 (39)
TopTaq 5 U/ul 0.05 0.425 5 AV10027 CC A3
Milli Q 5.75 48.875 6 AV10027 Pennsylvania (PA) D6 (71)
10 ul 7 AV10027 Baltimore B5 (72)
8 AV10027 Tsukuba3 D4 (39)
PCR cycle
94C 3 min
98C 10 sec
60C 3 min
x35
72C 5 min


load 2.5ul of sample

0091.jpg

QIAamp DNA mini extraction kit

Reaction mixture conc. 1x x16.5 ' lane E. Windsor lane Tsukuba3 D4
Temp (High molecular weight) 1ug/ul 2 1 AV10134 9 AV10134
TopTaq Buffer 10x 1 16.5 2 AV10121 10 AV10121
dNTP mix 10 mM each 0.2 3.3 3 AV10084 11 AV10084
Primer mix 5 uM each 1 4 AV10045 12 AV10045
TopTaq 5 U/ul 0.05 0.825 5 AV10044 13 AV10044
Milli Q 5.75 94.875 6 AV10034 14 AV10034
10 ul 7 AV10027 15 AV10027
8 AV10025 16 AV10025
PCR cycle
94C 3 min
98C 10 sec
60C 3 min
x35
72C 5 min

load 2.5ul of sample

0092.jpg

Gene-rich region amplification in CCA3

PCR

Reaction mixture conc. 1x 6
water 5.35 32.1
LA buffer 10x 1 6
dNTP mix 2.5 mM each 1.6 9.6
Primer mix 5 uM each 1
TakaraLA 5U/ul 0.05 0.3
template 3 ng/ul 1 6
9
  • standard long PCR conditions
avhsp-1_7-6 avhsp1_3_1_4 avhsp3_1-3 avhsp-7_1-5 avhsp-1_7-4 avhsp-1_7-3

Gel

Asm100420.jpg