Noi/NOTES/2012-5-25: Difference between revisions

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  '''Program'''<br>
  '''Program'''<br>
  98C 30s -> (98C 10s -> 58C 30s -> 72C 30s)x8 -> (98C 10s -> 72C 30s)x14 -> 72C 3min
  98C 30s -> (98C 10s -> 58C 30s -> 72C 30s)x8 -> (98C 10s -> 72C 30s)x14 -> 72C 3min
* I performed qPCR twice with the same conditions
* I performed qPCR in duplicate with the same condition and stopped at the same cycle
[[File:2012_05_25_qPCR_SR_plate1.png| 500px]] [[File:2012_05_25_qPCR_SR_plate2.png| 500px]]
[[File:2012_05_25_qPCR_SR_plate1.png| 500px]] [[File:2012_05_25_qPCR_SR_plate2.png| 500px]]
* 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

Revision as of 02:02, 31 May 2012

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 1 rxn 4.2 rxn mix
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
File:2012 05 25 testqPCR SR.png
  • 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 2,556.00
Total 100.00 6,248.00
  • Aliquot 88ul, add 2ul of 10uM AmpR6.3Indx and 10ul of circularized DNA template
Program
98C 30s -> (98C 10s -> 58C 30s -> 72C 30s)x8 -> (98C 10s -> 72C 30s)x14 -> 72C 3min
  • I performed qPCR in duplicate with the same condition and stopped at the same cycle

File:2012 05 25 qPCR SR plate1.png File:2012 05 25 qPCR SR plate2.png

  • 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 E8**
F8** 4.95 297.18 F8**
A9 8.36 501.40 A9
B9 7.97 478.09 B9
C9 8.74 524.33 C9
D9 7.70 461.86 D9
E9 6.27 376.26 E9
F9 7.92 474.91 F9
G9 4.52 270.94 G9
H9 7.51 450.74 H9
  • Note: For the sequencing libraries, NP-BSPP-SR_Ind1-60_May21,2012, I combined samples in well ID A1-D8 (total 60 libraries)
  • Performed PAGE size selection with 6% TBE 5-well gel (4 gels)
File:ZhangLab 2 2012-05-28 11hr 15min PAGE-SS-1.jpg File:ZhangLab 2 2012-05-28 11hr 17min PAGE-SS-2.jpg
  • Resuspended with H2O, total volume 60ul
  • Quantified concentration by Qubit dsDNA assay by Alan (based on size 375bp): 83.18nM or 14.4ng/ul
  • Concentration by PAGE quantification is 33.5ng/ul

File:ZhangLab 2 2012-05-30 17hr 20min PQ NP BSPP SR Ind1-60 May21 2012.jpg

  • Library IDs: NP-BSPP-SR_Ind1-60_May21,2012, sequenced by HiSeq at BioGem (Lane: 4-8)