Revision as of 18:44, 13 April 2009 by >Sam Chiang
Realtime PCR validation for L4N9 (3/31/'09) and N6 (04/01/'09) MDA amplicons
Procedure
- Experiment design
- Primers: 1-1, 2-1, 3-1, 4-1, 5-1, 6-1
- Templates: Candidates MDA amplicon. 7 samples from 3/31, 7 samples from 4/01, pos (gDNA 100pg/uL), NTC(H2O)
- Thaw gDNA(100pg/uL), Taq 2X enzyme, primers mix and diluted templates (MDA amplicon in 1/50 dilution)
- Set up realtime PCR program (decribed as following)
- Set up 12 PCR strip tubes on cool block, labed as column 1 to column 12.
Column 1 -> 6 Column 7 -> 12
Primer 1-1->6-1 Primer 1-1->6-1
B6(4-01) B7(4-01)
D2(4-01) D4(4-01)
D5(4-01) D7(4-01)
D8(4-01) B3(3-31)
B4(3-31) B6(3-31)
C2(3-31) C5(3-31)
C7(3-31) C8(3-31)
POS(100pg) NTC
- Prepare diluted primers in PCR tubes
1rxn (8+1)x2 ->20rxns
Primer mix (f+r 10 uM) 1.0 20.0 uL
H2O 7.0 140.0 uL
----------------------------------------
8.0 160.0
- Transfer 8 uL diluted primer into each reaction based on the arrangements
- Transfer 2 uL diluted templates into each reaction based on arrangements
- Prepare master mix in 1.5-mL microcentrifuge tube
1rxn (12+2)x8=112 rxns
Taq (2X) 9.9 1108.8
SYBR (50X) 0.1 11.2
--------------------------------
10.0 1120.0
- Mix well the master mix by vortexing. Seperate master into 8 PCR tubes then transer 10 uL Master mix into each reaction using multichannel pipettor.
- Gently vortex and spin down the PCR reaction tubes. Place in Chromo4 machine immediately to perform realtime PCR reaction.
- Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C sec -> plate read) x 45 -> Melting curve (52C->94C, read every 2C, hold 1min) -> 15C forever
Results
Primer 1-1
Content
|
Description
|
'
|
C(t)
|
'
|
dCT(NTC-Sample)
|
'
|
Melting curve matched to Pos control
|
Sample |
B6 (4/1) N6-No-Tre |
|
37.91 |
|
-1.06 |
|
n
|
Sample |
B7 (4/1) N6-No-Tre |
|
7.72 |
|
29.13 |
|
n
|
Sample |
D2 (4/1) N6-No-Tre |
|
22.07 |
|
14.78 |
|
n
|
Sample |
D4 (4/1) N6-No-Tre |
|
6.37 |
|
30.48 |
|
n
|
Sample |
D5 (4/1) N6-No-Tre |
|
6.77 |
|
30.08 |
|
n
|
Sample |
D7 (4/1) N6-No-Tre |
|
7.37 |
|
29.48 |
|
n
|
Sample |
D8 (4/1) N6-No-Tre |
|
5.48 |
|
31.37 |
|
n
|
Sample |
B3 (3/31) L4N9-Tre |
|
36.3 |
|
0.55 |
|
y
|
Sample |
B4 (3/31) L4N9-Tre |
|
33.25 |
|
3.6 |
|
y
|
Sample |
B6 (3/31) L4N9-Tre |
|
34.64 |
|
2.21 |
|
n
|
Sample |
C2 (3/31) L4N9-Tre |
|
30.49 |
|
6.36 |
|
y
|
Sample |
C5 (3/31) L4N9-Tre |
|
4.76 |
|
32.09 |
|
n
|
Sample |
C7 (3/31) L4N9-Tre |
|
31.67 |
|
5.18 |
|
y
|
Sample |
C8 (3/31) L4N9-Tre |
|
32.3 |
|
4.55 |
|
n
|
Sample |
pos |
|
24.3 |
|
|
|
|
Blank |
ntc |
|
36.85 |
|
|
|
|
|
Content
|
Description
|
C(t)
|
dCT(NTC-Sample)
|
Melting curve matched to Pos control
|
Sample |
B6 (4/1) N6-No-Tre |
37.91 |
-1.06 |
n
|
Sample |
B7 (4/1) N6-No-Tre |
7.72 |
29.13 |
n
|
Sample |
D2 (4/1) N6-No-Tre |
22.07 |
14.78 |
n
|
Sample |
D4 (4/1) N6-No-Tre |
6.37 |
30.48 |
n
|
Sample |
D5 (4/1) N6-No-Tre |
6.77 |
30.08 |
n
|
Sample |
D7 (4/1) N6-No-Tre |
7.37 |
29.48 |
n
|
Sample |
D8 (4/1) N6-No-Tre |
5.48 |
31.37 |
n
|
Sample |
B3 (3/31) L4N9-Tre |
36.3 |
0.55 |
y
|
Sample |
B4 (3/31) L4N9-Tre |
33.25 |
3.6 |
y
|
Sample |
B6 (3/31) L4N9-Tre |
34.64 |
2.21 |
n
|
Sample |
C2 (3/31) L4N9-Tre |
30.49 |
6.36 |
y
|
Sample |
C5 (3/31) L4N9-Tre |
4.76 |
32.09 |
n
|
Sample |
C7 (3/31) L4N9-Tre |
31.67 |
5.18 |
y
|
Sample |
C8 (3/31) L4N9-Tre |
32.3 |
4.55 |
n
|
Sample |
pos |
24.3 |
|
|
Blank |
ntc |
36.85 |
|
|
|
Content
|
Description
|
C(t)
|
dCT(NTC-Sample)
|
Melting curve matched to Pos control
|
Sample |
B6 (4/1) N6-No-Tre |
37.91 |
-1.06 |
n
|
Sample |
B7 (4/1) N6-No-Tre |
7.72 |
29.13 |
n
|
Sample |
D2 (4/1) N6-No-Tre |
22.07 |
14.78 |
n
|
Sample |
D4 (4/1) N6-No-Tre |
6.37 |
30.48 |
n
|
Sample |
D5 (4/1) N6-No-Tre |
6.77 |
30.08 |
n
|
Sample |
D7 (4/1) N6-No-Tre |
7.37 |
29.48 |
n
|
Sample |
D8 (4/1) N6-No-Tre |
5.48 |
31.37 |
n
|
Sample |
B3 (3/31) L4N9-Tre |
36.3 |
0.55 |
y
|
Sample |
B4 (3/31) L4N9-Tre |
33.25 |
3.60 |
y
|
Sample |
B6 (3/31) L4N9-Tre |
34.64 |
2.21 |
n
|
Sample |
C2 (3/31) L4N9-Tre |
30.49 |
6.36 |
y
|
Sample |
C5 (3/31) L4N9-Tre |
4.76 |
32.09 |
n
|
Sample |
C7 (3/31) L4N9-Tre |
31.67 |
5.18 |
y
|
Sample |
C8 (3/31) L4N9-Tre |
32.3 |
4.55 |
n
|
Sample |
pos |
24.3 |
|
|
Blank |
ntc |
36.85 |
|
|