User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/27

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Cycle Sequence AMPure Cleaned PCR Product

HCO primer only

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
Template * ? 4.6
126 1.4ul / RXN
* PEG 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

Nuclear Gene Rotifer Culture 454 Amplification

Adenita Culture 1-12

EX Takara RXN Mixture conc 1X 6.5 '
Milli Q water 5.15 33.475
Takara EX Buffer 10X 1 6.5
dNTP 2.5uM 0.8 5.2
Primer Mix 20uM 1
EX Taq 5U 0.05 0.325
Template Instagene 2 13
58.5 9.0ul / RXN
A PCR cycle
98C 10 sec
60C 1 min
x35
Plate 1
Source Plate Well EX ID
Baltimore A1 1
Baltimore B1 2
Baltimore C2 3
Baltimore C3 4
E. Windsor A1 5
E. Windsor A3 6
E. Windsor B1 7
E. Windsor B2 8
E. Windsor C2 9
Harvard A2 10
Harvard A3 11
Ithaca 1 A1 12