Noi/NOTES/2012-5-25: Difference between revisions
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* I performed qPCR twice with the same conditions | * 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 | * 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 | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Well ID''' | |||
| align="center" style="background:#f0f0f0;"|'''conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Total amount in 60ul (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume for 60ng (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''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) |
Revision as of 09:37, 28 May 2012
- [Link to calendar]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-21#SLN_mix_solution
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)