Adenita Culture 454 Amplification Adapter extension
EX Takara RXN Mixture
|
conc
|
1X
|
34
|
Milli Q water |
|
6.15 |
209.1
|
Takara EX Buffer |
10X |
1 |
34
|
dNTP |
2.5uM |
0.8 |
27.2
|
Primer Mix |
20uM |
1 |
|
EX Taq |
5U |
0.05 |
1.7
|
Template |
Oleg PCR 1:10 |
1 |
|
|
|
|
272
|
|
|
|
8.0ul / RXN add 1.0ul Primer and 1.0ul PCR template
|
|
|
|
|
|
|
|
|
PCR cycle |
|
|
|
98C |
10 sec |
|
|
60C |
1 min |
|
|
x15 |
|
|
|
Primer
|
Colonies ----->
|
11
|
27
|
29
|
32
|
40
|
41
|
43
|
50
|
54
|
55
|
56
|
D |
HSP82 F2/R2 |
ok =1 |
ok = 5 |
ok = 7 |
ok = 10 |
ok = 13 |
ok = 16 |
ok = 19 |
? = 22 |
ok = 25 |
ok = 28 |
ok = 31
|
B |
Hist F1/H |
ok =2 |
failed |
ok = 8 |
ok = 11 |
ok = 14 |
failed |
ok = 20 |
ok = 23 |
ok = 26 |
ok = 29 |
? = 32
|
E |
Myosin F2/R2 |
ok =3 |
failed |
failed |
failed |
failed |
failed |
failed |
failed |
ok = 27 |
failed |
failed
|
F |
Rho |
ok =4 |
ok = 6 |
ok = 9 |
ok = 12 |
ok =15 |
ok = 17 |
ok = 21 |
ok = 24 |
failed |
ok = 30 |
ok = 33
|
C |
Histone 2 |
|
failed |
failed |
failed |
failed |
ok = 18 |
failed |
failed |
failed |
failed |
failed
|
Primer Set |
|
11 |
3 |
5 |
8 |
4 |
5 |
7 |
2 |
6 |
7 |
8
|
Loaded 2.0ul
Library preparation for Alien genes
Library amplification
Reaction mixture
|
conc.
|
1x
|
template (after adapter ligation 1 day liagtion) |
33.8ng/ul |
2.5
|
Atitin primer |
10uM |
0.5
|
Btitin primer |
10uM |
0.5
|
Phusion DNA polymerase |
2x |
12.5
|
MilliQ |
|
9
|
|
total |
25
|
|
|
|
98C |
30 sec |
|
|
|
|
98C |
10 sec |
|
65C |
30 sec |
|
72C |
30 sec |
|
|
25 cycle |
|
|
|
|
72C |
5 min |
|
|
|
|
load 1.0 ul of sample
|