Revision as of 15:27, 28 May 2012 by >Noi
PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX No.1-72)
- I tested if the capture works well by performing qPCR of few samples in small volume 25ul, sample E1, F1, G1 and NTC
Captured template
|
2.5
|
0
|
10uM AmpF6.4Sol |
0.5 |
2.1
|
10uM AmpR6.3Sol |
0.5 |
2.1
|
50x SYBG |
0.2 |
0.84
|
2x Phusion MM |
12.5 |
52.5
|
H2O |
8.8 |
36.96
|
- Aliquot 22.5ul to each tube and add 2.5ul of circularized DNA templates
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
- Form the qPCR of tested samples, I could observe amplification in every samples except NTC, so I can continue amplify all samples and barcode with index primers.
- I have to aliquot AmpR3.3Indx primer due to the evaporation of the old plate storing at 4C. In the future I might store at -20C
Plate layout and barcoded primer
A1
|
A2
|
A3
|
A4
|
A5
|
A6
|
A7
|
A8
|
A9
|
A10
|
A11
|
A12
|
AmpR6.3.Ind1 |
AmpR6.3.Ind9 |
AmpR6.3.Ind17 |
AmpR6.3.Ind25 |
AmpR6.3.Ind33 |
AmpR6.3.Ind41 |
AmpR6.3.Ind49 |
AmpR6.3.Ind57 |
AmpR6.3.Ind65 |
AmpR6.3.Ind73 |
AmpR6.3.Ind81 |
AmpR6.3.Ind89
|
B1 |
B2 |
B3 |
B4 |
B5 |
B6 |
B7 |
B8 |
B9 |
B10 |
B11 |
B12
|
AmpR6.3.Ind2 |
AmpR6.3.Ind10 |
AmpR6.3.Ind18 |
AmpR6.3.Ind26 |
AmpR6.3.Ind34 |
AmpR6.3.Ind42 |
AmpR6.3.Ind50 |
AmpR6.3.Ind58 |
AmpR6.3.Ind66 |
AmpR6.3.Ind74 |
AmpR6.3.Ind82 |
AmpR6.3.Ind90
|
C1 |
C2 |
C3 |
C4 |
C5 |
C6 |
C7 |
C8 |
C9 |
C10 |
C11 |
C12
|
AmpR6.3.Ind3 |
AmpR6.3.Ind11 |
AmpR6.3.Ind19 |
AmpR6.3.Ind27 |
AmpR6.3.Ind35 |
AmpR6.3.Ind43 |
AmpR6.3.Ind51 |
AmpR6.3.Ind59 |
AmpR6.3.Ind67 |
AmpR6.3.Ind75 |
AmpR6.3.Ind83 |
AmpR6.3.Ind91
|
D1 |
D2 |
D3 |
D4 |
D5 |
D6 |
D7 |
D8 |
D9 |
D10 |
D11 |
D12
|
AmpR6.3.Ind4 |
AmpR6.3.Ind12 |
AmpR6.3.Ind20 |
AmpR6.3.Ind28 |
AmpR6.3.Ind36 |
AmpR6.3.Ind44 |
AmpR6.3.Ind52 |
AmpR6.3.Ind60 |
AmpR6.3.Ind68 |
AmpR6.3.Ind76 |
AmpR6.3.Ind84 |
AmpR6.3.Ind92
|
E1 |
E2 |
E3 |
E4 |
E5 |
E6 |
E7 |
E8 |
E9 |
E10 |
E11 |
E12
|
AmpR6.3.Ind5 |
AmpR6.3.Ind13 |
AmpR6.3.Ind21 |
AmpR6.3.Ind29 |
AmpR6.3.Ind37 |
AmpR6.3.Ind45 |
AmpR6.3.Ind53 |
AmpR6.3.Ind61 |
AmpR6.3.Ind69 |
AmpR6.3.Ind77 |
AmpR6.3.Ind85 |
AmpR6.3.Ind93
|
F1 |
F2 |
F3 |
F4 |
F5 |
F6 |
F7 |
F8 |
F9 |
F10 |
F11 |
F12
|
AmpR6.3.Ind6 |
AmpR6.3.Ind14 |
AmpR6.3.Ind22 |
AmpR6.3.Ind30 |
AmpR6.3.Ind38 |
AmpR6.3.Ind46 |
AmpR6.3.Ind54 |
AmpR6.3.Ind62 |
AmpR6.3.Ind70 |
AmpR6.3.Ind78 |
AmpR6.3.Ind86 |
AmpR6.3.Ind94
|
G1 |
G2 |
G3 |
G4 |
G5 |
G6 |
G7 |
G8 |
G9 |
G10 |
G11 |
G12
|
AmpR6.3.Ind7 |
AmpR6.3.Ind15 |
AmpR6.3.Ind23 |
AmpR6.3.Ind31 |
AmpR6.3.Ind39 |
AmpR6.3.Ind47 |
AmpR6.3.Ind55 |
AmpR6.3.Ind63 |
AmpR6.3.Ind71 |
AmpR6.3.Ind79 |
AmpR6.3.Ind87 |
AmpR6.3.Ind95
|
H1 |
H2 |
H3 |
H4 |
H5 |
H6 |
H7 |
H8 |
H9 |
H10 |
H11 |
H12
|
AmpR6.3.Ind8 |
AmpR6.3.Ind16 |
AmpR6.3.Ind24 |
AmpR6.3.Ind32 |
AmpR6.3.Ind40 |
AmpR6.3.Ind48 |
AmpR6.3.Ind56 |
AmpR6.3.Ind64 |
AmpR6.3.Ind72 |
AmpR6.3.Ind80 |
AmpR6.3.Ind88 |
AmpR6.3.Ind96
|
- Note: There are total 70 reactions (A1-H9, no samples in well ID G8 and H8 and A10-H12)
Components
|
1x rxn
|
71 rxn mix
|
Captured template |
10.00 |
0.00
|
10uM AmpF6.4Sol |
2.00 |
142.00
|
10uM AmpR6.3Indx (X=1-72) |
2.00 |
0.00
|
2x Kapa SYBR MM |
50.00 |
3550.00
|
H2O |
36.00 |
2556.00
|
Total |
100.00 |
6248.00
|
Program
98C 30s -> (98C 10s -> 58C 30s -> 72C 30s)x8 -> (98C 10s -> 72C 30s)x14 -> 72C 3min
- I performed qPCR twice with the same conditions
- Since A9-H9 reactions shoot up earlier than other samples, I took out 60ul at cycles 20
- Took 60ul of PCR product from plate1 and 2 (total volume 120ul)
- Purified with 0.8 vol of AmPure beads
- Eluted with 60ul EB buffer
- Performed PAGE quantification by loading sample 1ul
Well ID
|
conc. (ng/ul)
|
Total amount in 60ul (ng)
|
Volume for 60ng (ul)
|
Well ID
|
A1 |
13.66 |
819.34 |
4.39 |
A1
|
B1 |
11.97 |
718.02 |
5.01 |
B1
|
C1 |
12.50 |
750.04 |
4.80 |
C1
|
D1 |
11.37 |
682.37 |
5.28 |
D1
|
E1 |
9.32 |
559.04 |
6.44 |
E1
|
F1 |
11.67 |
699.95 |
5.14 |
F1
|
G1 |
12.32 |
739.22 |
4.87 |
G1
|
H1 |
13.97 |
838.08 |
4.30 |
H1
|
A2 |
14.19 |
851.55 |
4.23 |
A2
|
B2 |
13.07 |
784.08 |
4.59 |
B2
|
C2 |
14.71 |
882.63 |
4.08 |
C2
|
D2 |
14.17 |
850.27 |
4.23 |
D2
|
E2 |
17.12 |
1026.97 |
3.51 |
E2
|
F2 |
16.72 |
1003.03 |
3.59 |
F2
|
G2 |
17.42 |
1045.27 |
3.44 |
G2
|
H2* |
15.41 |
924.62 |
3.89 |
H2*
|
A3 |
14.31 |
858.62 |
4.19 |
A3
|
B3 |
16.01 |
960.41 |
3.75 |
B3
|
C3 |
15.83 |
949.64 |
3.79 |
C3
|
D3 |
10.69 |
641.17 |
5.61 |
D3
|
E3 |
6.92 |
415.03 |
8.67 |
E3
|
F3 |
12.93 |
775.83 |
4.64 |
F3
|
G3 |
12.04 |
722.32 |
4.98 |
G3
|
H3 |
12.10 |
726.03 |
4.96 |
H3
|
A4 |
16.39 |
983.11 |
3.66 |
A4
|
B4 |
15.02 |
901.02 |
4.00 |
B4
|
C4 |
12.66 |
759.53 |
4.74 |
C4
|
D4 |
9.03 |
541.89 |
6.64 |
D4
|
E4 |
16.28 |
976.88 |
3.69 |
E4
|
F4 |
11.63 |
698.04 |
5.16 |
F4
|
G4* |
8.79 |
527.68 |
6.82 |
G4*
|
H4 |
17.56 |
1053.63 |
3.42 |
H4
|
A5* |
11.98 |
718.73 |
5.01 |
A5*
|
B5* |
12.10 |
726.10 |
4.96 |
B5*
|
C5 |
13.53 |
811.88 |
4.43 |
C5
|
D5 |
14.28 |
856.59 |
4.20 |
D5
|
E5 |
18.77 |
1125.92 |
3.20 |
E5
|
F5 |
13.74 |
824.13 |
4.37 |
F5
|
G5 |
15.84 |
950.23 |
3.79 |
G5
|
H5 |
18.66 |
1119.88 |
3.21 |
H5
|
A6 |
14.10 |
845.88 |
4.26 |
A6
|
B6 |
15.23 |
913.85 |
3.94 |
B6
|
C6 |
15.54 |
932.41 |
3.86 |
C6
|
D6 |
14.23 |
853.91 |
4.22 |
D6
|
E6 |
13.29 |
797.58 |
4.51 |
E6
|
F6 |
13.74 |
824.66 |
4.37 |
F6
|
G6 |
14.29 |
857.67 |
4.20 |
G6
|
H6 |
13.94 |
836.22 |
4.31 |
H6
|
A7 |
15.95 |
956.98 |
3.76 |
A7
|
B7 |
14.68 |
880.72 |
4.09 |
B7
|
C7 |
14.81 |
888.76 |
4.05 |
C7
|
D7 |
15.94 |
956.39 |
3.76 |
D7
|
E7* |
6.96 |
417.31 |
8.63 |
E7*
|
F7** |
4.17 |
250.11 |
14.39 |
F7**
|
G7 |
15.68 |
940.75 |
3.83 |
G7
|
H7* |
13.03 |
781.77 |
4.60 |
H7*
|
A8 |
15.51 |
930.69 |
3.87 |
A8
|
B8 |
14.18 |
850.94 |
4.23 |
B8
|
C8* |
12.09 |
725.59 |
4.96 |
C8*
|
D8 |
11.08 |
664.88 |
5.41 |
D8
|
E8** |
2.68 |
160.74 |
22.40 |
E8**
|
F8** |
4.95 |
297.18 |
12.11 |
F8**
|
A9 |
8.36 |
501.40 |
7.18 |
A9
|
B9 |
7.97 |
478.09 |
7.53 |
B9
|
C9 |
8.74 |
524.33 |
6.87 |
C9
|
D9 |
7.70 |
461.86 |
7.79 |
D9
|
E9 |
6.27 |
376.26 |
9.57 |
E9
|
F9 |
7.92 |
474.91 |
7.58 |
F9
|
G9 |
4.52 |
270.94 |
13.29 |
G9
|
H9 |
7.51 |
450.74 |
7.99 |
H9
|
- Note: I will include sample A1-D8 for the pooled sequencing libraries (total 60 libraries)
- Library IDs: NP-BSPP-SR_Ind1-60_May21,2012