User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/01

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Contents

LCO HCO

Amplification of HCO/LCO on cultures


Reaction mixture conc. 1x 19 '
Template Instagene?1
TopTaq buffer10x119
dNTP mix10 mM each0.23.8
Primer mix5 uM each119
TopTaq (QIAGEN)5U/ul0.050.95
Milli Q water6.75128.25
10 ul1719.0ul / RXN
*Control AV 1ng/ul
PCR cycle
94C3 min
94C10 sec
45-68C3 min
RAMP 0.5C/sec
x35
68C5 min
AMP Lane Template ' Sample Number
1Ithaca 1C648
2HarvardA449
3Ithaca 1B450
4Ithaca 1C551
5BaltimoreB252
6BaltimoreB353
7BaltimoreB654
8E. WindsorC355
9Pennsylvania (PA)D656
10BaltimoreB557
11Ithaca 1C258
12Pennsylvania (PA)D559
13Woods Hole 1B3A
14Woods Hole 1B5B
15Woods Hole 1C4C
16Woods Hole 2A6D
17Positive Control AV
18Negative Control

Load 1.0ul sample image:01april10.jpg

Alien Gene LongPCR

Amplification of TEL_L_B

' [CONC] 1X 12 ' ' AMP Lane Template MIX Contents ' Size Fosmid
Water5.363.61AV134AV10098 FWDAV10104 FWDAV TEL L B
Takara LA Taq5U0.11.22AV135AV10092 FWDAV10093 RVSAV TEL L B
Takara LA Buffer10X1123AV136AV10092 FWDAV10093.2 RVSAV TEL L B
dNTP2.5uM each1.619.24CCA3134AV10098 FWDAV10108.2 RVSAV TEL L B
*Template1ng1125CCA3135AV10092 FWDAV10093 RVSAV TEL L B
Primer Mix5uM each16CCA3136AV10092 FWDAV10093.2 RVSAV TEL L B
10.0ul1089.0ul/RXN7CCA384AV10092.2 FWDAV10093.2 RVSAV TEL L B
add 1.0 Primer Mix8CCA385AV10092.2 FWDAV10094 RVSAV TEL L B
* AV or CCA3
PCR Cycle
94C 1 min
98C10 sec
65C15 min
30X

Amplification of TEL_L_A new primers on CCA3

Reaction mixture conc. 1x 5 ' AMP Lane Template Contents
*Template 1ng/ul15AAVAV10083
TopTaq buffer10x15BAVAV10084.2
dNTP mix10 mM each0.21CAVAV10085
Primer mix5 uM each1DAVAV10090
TopTaq (QIAGEN)5U/ul0.050.25ECCA3AV10083
Milli Q water6.7533.75FCCA3AV10084.2
10 ul45GCCA3AV10085
*CCA3 or AV9.0ul/RXNHCCA3AV10090
PCR cycle
94C3 min
98C10 sec
60C3 min
x35
72C5 min


Lanes 1-6 Load 1.0ul sample

Lanes 7,8 & A-H Load 2.0ul sample

image:01apr10 gel2.jpg

Alien Gene LongPCR 2

Amplification of TEL_L_B

' [CONC] 1X 12 ' ' AMP Lane Template MIX Contents ' Size Fosmid
Water5.363.61AV123AV10108 FWDAV10108.2 RVSAV TEL L B
Takara LA Taq5U0.11.22AV124TEL_L_B_2 FWDTEL_L_B_3 RVSAV TEL L B
Takara LA Buffer10X1123AV125TEL_L_B_2 FWDTEL_L_B_4 RVSAV TEL L B
dNTP2.5uM each1.619.24AV126TEL_L_B_3 FWDTEL_L_B_4 RVSAV TEL L B
*Template1ng1125AV127AV10113 FWDTEL_L_B_2 RVSAV TEL L B
Primer Mix5uM each16AV128AV10113 FWDTEL_L_B_3 RVSAV TEL L B
10.0ul1089.0ul/RXN7AV129AV10113 FWDTEL_L_B_4 RVSAV TEL L B
add 1.0 Primer Mix8AV130TEL_L_B_3 FWDAV10114 RVSAV TEL L B
9AV131AV10114 FWDTEL_L_B_4 RVSAV TEL L B
* AV or CCA310AV132TEL_L_B_4 FWDAV10114 RVSAV TEL L B
11AV133TEL_L_B_4 FWDTEL_L_B_3 RVSAV TEL L B
PCR Cycle12CCA3123AV10108 FWDAV10108.2 RVSAV TEL L B
94C 1 min13CCA3124TEL_L_B_2 FWDTEL_L_B_3 RVSAV TEL L B
98C10 sec14CCA3125TEL_L_B_2 FWDTEL_L_B_4 RVSAV TEL L B
65C15 min15CCA3126TEL_L_B_3 FWDTEL_L_B_4 RVSAV TEL L B
30X16CCA3127AV10113 FWDTEL_L_B_2 RVSAV TEL L B
17CCA3128AV10113 FWDTEL_L_B_3 RVSAV TEL L B
18CCA3129AV10113 FWDTEL_L_B_4 RVSAV TEL L B
19CCA3130TEL_L_B_3 FWDAV10114 RVSAV TEL L B
20CCA3131AV10114 FWDTEL_L_B_4 RVSAV TEL L B
21CCA3132TEL_L_B_4 FWDAV10114 RVSAV TEL L B
22CCA3133TEL_L_B_4 FWDTEL_L_B_3 RVSAV TEL L B

Load 1.0ul of sample

image:01apr10 gel1 .jpg

Alien Genes New Primer Screen

Reaction mixture conc. 1x x16.5 ' lane '
Temp??21AV10134
TopTaq Buffer10x116.52AV10121
dNTP mix10 mM each0.23.33AV10084
Primer mix (HIS F/R1)5 uM each116.54AV10045
TopTaq5 U/ul0.050.8255AV10044
Milli Q5.7594.8756AV10034
10 ul7AV10027
8AV10025
PCR cycle
94C3 minsample
61Pennsylvania (PA) B3
98C10 sec62Ithaca 1 C3
60C3 min
x35
72C5 min

image:0058.jpg

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