Noi/NOTES/2012-5-25: Difference between revisions
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| 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 | | 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}} | * Note: There are total 70 reactions (A1-H9, no samples in well ID G8 and H8 and A10-H12) | ||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''1x rxn''' | | align="center" style="background:#f0f0f0;"|'''1x rxn''' | ||
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| 10uM AmpR6.3Indx (X=1-72)||2.00||0.00 | | 10uM AmpR6.3Indx (X=1-72)||2.00||0.00 | ||
|- | |- | ||
| 2x Kapa MM||50.00||3550.00 | | 2x Kapa SYBR MM||50.00||3550.00 | ||
|- | |- | ||
| H2O||36.00||2556.00 | | H2O||36.00||2556.00 | ||
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|} | |} | ||
'''Program'''<br> | '''Program'''<br> | ||
98C 30s -> (98C 10s -> 58C 30s -> 72C 30s)x8 -> (98C 10s -> 72C 30s) | 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 |
Revision as of 08:25, 26 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 |
- 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