Noi/NOTES/2012-5-25: Difference between revisions
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98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min | 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. | * 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 === | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''A1''' | |||
| align="center" style="background:#f0f0f0;"|'''A2''' | |||
| align="center" style="background:#f0f0f0;"|'''A3''' | |||
| align="center" style="background:#f0f0f0;"|'''A4''' | |||
| align="center" style="background:#f0f0f0;"|'''A5''' | |||
| align="center" style="background:#f0f0f0;"|'''A6''' | |||
| align="center" style="background:#f0f0f0;"|'''A7''' | |||
| align="center" style="background:#f0f0f0;"|'''A8''' | |||
| align="center" style="background:#f0f0f0;"|'''A9''' | |||
| align="center" style="background:#f0f0f0;"|'''A10''' | |||
| align="center" style="background:#f0f0f0;"|'''A11''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''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 MM||50.00||3550.00 | |||
|- | |||
| H2O||36.00||2556.00 | |||
|- | |||
| Total||100.00||6248.00 | |||
|} | |||
'''Program'''<br> | |||
98C 30s -> (98C 10s -> 58C 30s -> 72C 30s)x8 -> (98C 10s -> 72C 30s)x15 -> 72C 3min |
Revision as of 23:56, 25 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-)
- 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 |
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 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)x15 -> 72C 3min