#04-02 qPCR 96well evenness test 4/2/14 1:19 PM

Wednesday, March 19, 2014 8:52 PM

Part 1

EXPERIMENT TARGET:

Evenness test of Bio-Rad CFX-96 qPCR SYSTEM

Sample Preparation:

Pro 9312 culture: 1e-1 1μl 25x

Part 2

No. COMPONENT SOURCE STOCK 1x (25μl) FINAL X25 Notes

1. Premix Taq Tiangen NA 12.5 NA 312.5
2. Forw. Primer 9312_276f 10μM 0.5 0.2μM 12.5
3. Reve. Primer 9312_276r 10μM 0.5 0.2μM 12.5
4. ddWater Tiangen NA 10.5 NA 262.5
5. Sample Specify 1x 1 NA 25
Other

Part 3

qPCR Vol.= 12.5μl

No. 1 2 3 4 5 6 7 8
Step Pre Heat. Heat. Anneal. Elong. FL. Dete. Go to 2 Final Elong. Melt. Cur
Tem.℃ 95 95 62 72 79 Na Na 60-95
Time 15 min 30s 30s 30s 15s 40 cycles 5min 5s
Note
 9
rv. Preserve
Na
Forever
Tem.℃ 95 95 62 72 79 Na Na 60-95
Time 15 min 30s 30s 30s 15s 40 cycles 5min 5s
Note

Part 4

qPCR 96 well plate settings

Tuesday, March 18, 2014
5:32 PM
Set 1 2 3 4 5 6 7 8 9
A 12.5 12.5 12.5 12.5
B 12.5 12.5 12.5 12.5
C 12.5 12.5 12.5 12.5
D 12.5 12.5 12.5 12.5
E 12.5 12.5 12.5 12.5
F 12.5 12.5 12.5 12.5
G 12.5 12.5 na 12.5
H 12.5 12.5 na na

Part 5
qPCR 96 well plate results Bio-Rad Files : 2014-4-2
Res 1 2 3 4 5 6 7 8 9
A 19.04 19.06 18.89 19.22
B 18.51 18.85 19.13 19.03
C 18.96 19.22 19.19 18.88
D 18.70 19.10 19.41 19.02
E 18.88 19.16 19.00 18.93
F 18.83 19.09 18.96 18.96
G 19.01 19.08 NA 19.22
H 19.03 19.17 NA NA
Note: C12 has significant difference!
 Na
Forever

10 11 12
12.5 12.5
12.5 12.5
12.5 12.5
12.5 12.5
12.5 12.5
12.5 12.5
12.5 12.5
12.5 12.5

10 11 12
19.00 18.98
18.99 18.61
19.17 18.88
19.15 18.44
18.82 18.71
19.04 18.70
19.03 19.14
19.04 18.54
G 19.01 19.08 NA 19.22
H 19.03 19.17 NA NA
Note: C12 has significant difference!

96 well
eveness test
19.03 19.14
19.04 18.54