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

From ZhangLabWiki
Jump to navigation Jump to search
>Noi
>Noi
No edit summary
 
(3 intermediate revisions by the same user not shown)
Line 248: Line 248:
[[File:ZhangLab_2 2012-05-30 17hr 20min_PQ_NP_BSPP_SR_Ind1-60_May21_2012.jpg| 180px]]
[[File:ZhangLab_2 2012-05-30 17hr 20min_PQ_NP_BSPP_SR_Ind1-60_May21_2012.jpg| 180px]]
* '''Library IDs: NP-BSPP-SR_Ind1-60_May21,2012''', sequenced on '''HiseqMay29''' run at BioGem (Lane: 4-8)
* '''Library IDs: NP-BSPP-SR_Ind1-60_May21,2012''', sequenced on '''HiseqMay29''' run at BioGem (Lane: 4-8)
** '''Note: Mapping was performed by Dinh'''.
** '''Raw reads: /media/SeqStore2/120531_SN100_DD/NP_BSPP'''
* The rest of libraries (Ind61,62,65-72) were pooled in another library, '''NP_BSPP_SR_HV_Ind61.62.65-80_May31,2012:''' continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-31
* The rest of libraries (Ind61,62,65-72) were pooled in another library, '''NP_BSPP_SR_HV_Ind61.62.65-80_May31,2012:''' continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-31
 
* Update: Library IDs: '''NP_BSPP_SR_HV_Ind61.62.65-80_May31,2012''', sequenced on '''HiSeqJun7''' run at Biogem ('''Lane: 8''')
== Barcodes ==
== Sample indexes ==
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Sample IDs'''
| align="center" style="background:#f0f0f0;"|'''Sample IDs'''

Latest revision as of 22:47, 18 June 2012

PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX No.1-72)[edit]

  • 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[edit]

A1 A2 A3 A4 A5 A6 A7 A8
AmpR6.3.Ind1 AmpR6.3.Ind9 AmpR6.3.Ind17 AmpR6.3.Ind25 AmpR6.3.Ind33 AmpR6.3.Ind41 AmpR6.3.Ind49 AmpR6.3.Ind57
B1 B2 B3 B4 B5 B6 B7 B8
AmpR6.3.Ind2 AmpR6.3.Ind10 AmpR6.3.Ind18 AmpR6.3.Ind26 AmpR6.3.Ind34 AmpR6.3.Ind42 AmpR6.3.Ind50 AmpR6.3.Ind58
C1 C2 C3 C4 C5 C6 C7 C8
AmpR6.3.Ind3 AmpR6.3.Ind11 AmpR6.3.Ind19 AmpR6.3.Ind27 AmpR6.3.Ind35 AmpR6.3.Ind43 AmpR6.3.Ind51 AmpR6.3.Ind59
D1 D2 D3 D4 D5 D6 D7 D8
AmpR6.3.Ind4 AmpR6.3.Ind12 AmpR6.3.Ind20 AmpR6.3.Ind28 AmpR6.3.Ind36 AmpR6.3.Ind44 AmpR6.3.Ind52 AmpR6.3.Ind60
E1 E2 E3 E4 E5 E6 E7 E8
AmpR6.3.Ind5 AmpR6.3.Ind13 AmpR6.3.Ind21 AmpR6.3.Ind29 AmpR6.3.Ind37 AmpR6.3.Ind45 AmpR6.3.Ind53 AmpR6.3.Ind61
F1 F2 F3 F4 F5 F6 F7 F8
AmpR6.3.Ind6 AmpR6.3.Ind14 AmpR6.3.Ind22 AmpR6.3.Ind30 AmpR6.3.Ind38 AmpR6.3.Ind46 AmpR6.3.Ind54 AmpR6.3.Ind62
G1 G2 G3 G4 G5 G6 G7
AmpR6.3.Ind7 AmpR6.3.Ind15 AmpR6.3.Ind23 AmpR6.3.Ind31 AmpR6.3.Ind39 AmpR6.3.Ind47 AmpR6.3.Ind55
H1 H2 H3 H4 H5 H6 H7
AmpR6.3.Ind8 AmpR6.3.Ind16 AmpR6.3.Ind24 AmpR6.3.Ind32 AmpR6.3.Ind40 AmpR6.3.Ind48 AmpR6.3.Ind56
  • 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 on HiseqMay29 run at BioGem (Lane: 4-8)
    • Note: Mapping was performed by Dinh.
    • Raw reads: /media/SeqStore2/120531_SN100_DD/NP_BSPP
  • The rest of libraries (Ind61,62,65-72) were pooled in another library, NP_BSPP_SR_HV_Ind61.62.65-80_May31,2012: continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-31
  • Update: Library IDs: NP_BSPP_SR_HV_Ind61.62.65-80_May31,2012, sequenced on HiSeqJun7 run at Biogem (Lane: 8)

Sample indexes[edit]

Sample IDs Lab IDs Well IDs IndX
Cell pellet or gDNA samples from Sergio
HUES2 p41 Ap15-1 A1 AmpR6.3.Ind1
ASThiPS4F1 p12 Ap15-2 B1 AmpR6.3.Ind2
ASThiPS4F2 p12 Ap15-3 C1 AmpR6.3.Ind3
HUES6 p39 Control TeSR Ap15-4 D1 AmpR6.3.Ind4
HUES6 p39 ACTIVIN Ap15-5 E1 AmpR6.3.Ind5
HUES6 p39 BMP-4 Ap15-6 F1 AmpR6.3.Ind6
HUES9 p39 Control TeSR Ap15-7 G1 AmpR6.3.Ind7
HUES9 p39 ACTIVIN Ap15-8 H1 AmpR6.3.Ind8
HUES9 p39 BMP-4 Ap15-9 A2 AmpR6.3.Ind9
HUES3 p36 Control TeSR Ap15-10 B2 AmpR6.3.Ind10
HUES3 p36 ACTIVIN Ap15-11 C2 AmpR6.3.Ind11
HUES3 p36 BMP-4 Ap15-12 D2 AmpR6.3.Ind12
H1 p54 Control TeSR Ap15-13 E2 AmpR6.3.Ind13
H1 p54 ACTIVIN Ap15-14 F2 AmpR6.3.Ind14
ASThiPS4F5 p16 Control TeSR Ap15-15 G2 AmpR6.3.Ind15
ASThiPS4F5 p16 ACTIVIN Ap15-16 H2 AmpR6.3.Ind16
ASThiPS4F4 p36 Control TeSR Ap15-17 A3 AmpR6.3.Ind17
ASThiPS4F4 p36 ACTIVIN Ap15-18 B3 AmpR6.3.Ind18
ASThiPS4F4 p36 BMP-4 Ap15-19 C3 AmpR6.3.Ind19
Huv-iPS4F6 p23 ACTIVIN Ma15-1 D3 AmpR6.3.Ind20
H1 p48 Ma15-2 E3 AmpR6.3.Ind21
H9 p47 Ma15-3 F3 AmpR6.3.Ind22
HUES3 p33 Ma15-4 G3 AmpR6.3.Ind23
HUES6 p26 Ma15-5 H3 AmpR6.3.Ind24
HUES8 p35 Ma15-6 A4 AmpR6.3.Ind25
HUES9 p36 Ma15-7 B4 AmpR6.3.Ind26
ASThiPS4F3 p24 (derived from Astrocytes) Ma15-8 C4 AmpR6.3.Ind27
ASThiPS4F4 p16 (derived from Astrocytes) Ma15-9 D4 AmpR6.3.Ind28
FiPS4F2 p32 (derived from IMR90 fibroblasts) Ma15-10 E4 AmpR6.3.Ind29
FiPS4F5 p29 (derived from IMR90 fibroblasts) Ma15-11 F4 AmpR6.3.Ind30
KiPS4FA p54 (derived from keratinocytes) Ma15-12 G4 AmpR6.3.Ind31
Huv-iPS4F6 p23 (derived from HUVECs) Ma15-13 H4 AmpR6.3.Ind32
IMR90 p6 Ma15-14 A5 AmpR6.3.Ind33
Keratinocytes p3 Ma15-15 B5 AmpR6.3.Ind34
H1 p47 Control Ma15-16 C5 AmpR6.3.Ind35
H1 p47 BMP-4 Ma15-17 D5 AmpR6.3.Ind36
ASThiPS4F5 p12 Control Ma15-18 E5 AmpR6.3.Ind37
ASThiPS4F5 p12 BMP-4 Ma15-19 F5 AmpR6.3.Ind38
Huv-iPS4F6 p23 Control Ma15-20 G5 AmpR6.3.Ind39
Huv-iPS4F6 p23 BMP-4 Ma15-21 H5 AmpR6.3.Ind40
H9 p43 mTeSR SR-1 A6 AmpR6.3.Ind41
H9 p43 ACTIVIN SR-2 B6 AmpR6.3.Ind42
H9 p43 BMP4 SR-3 C6 AmpR6.3.Ind43
HUV0iPS4F1 p19 SR-4 D6 AmpR6.3.Ind44
HUV-hiPS4F1 p20 ACTIVIN SR-5 E6 AmpR6.3.Ind45
HUV-hiPS4F1 p20 BMP4 SR-6 F6 AmpR6.3.Ind46
HUV-iPS4F3 F19 SR-7 G6 AmpR6.3.Ind47
HUV-hiPS4F3 p20 ACTIVIN SR-8 H6 AmpR6.3.Ind48
HUV-hiPS4F3 p20 BMP4 SR-9 A7 AmpR6.3.Ind49
aMSCs p7 SR-10 B7 AmpR6.3.Ind50
MSCiPS #8 p14 SR-11 C7 AmpR6.3.Ind51
MSCiPS #4 p14 SR-12 D7 AmpR6.3.Ind52
hNiPS2F p23 SR-13 E7 AmpR6.3.Ind53
hKiPS3F-7 p24 SR-14 F7 AmpR6.3.Ind54
hNSC SR-15 G7 AmpR6.3.Ind55
hFiPS4F-7 p9 SR-16 H7 AmpR6.3.Ind56
HUVEC p2 SR-17 A8 AmpR6.3.Ind57
hiPS AST4F-4 p9 SR-18 B8 AmpR6.3.Ind58
KERAT MMTA SR-19 C8 AmpR6.3.Ind59
KiPS4F #8 SR-20 D8 AmpR6.3.Ind60
olf MSC SR-21 E8 AmpR6.3.Ind61
KiPS SR-22 F8 AmpR6.3.Ind62
Old bis-cvt gDNA by Dinh (09/01/2010)
Astrocytes D10 SRB_1 A9 AmpR6.3.Ind65
AstiPS4F4 D11 SRB_2 B9 AmpR6.3.Ind66
AstiPS4F5 D9 SRB_3 C9 AmpR6.3.Ind67
HUVEC D12 SRB_4 D9 AmpR6.3.Ind68
HUViPS4F1 D13 SRB_5 E9 AmpR6.3.Ind69
HUViPS4F3 D14 SRB_6 F9 AmpR6.3.Ind70
HFF-XF D3 SRB_7 G9 AmpR6.3.Ind71
hNSC D1 SRB_8 H9 AmpR6.3.Ind72
gDNA samples from Harvard (ASM ASCM project)
GM gDNA 4-5-12 GM AmpR6.3.Ind73
P10E3 gDNA 4-5-12 P10E3 AmpR6.3.Ind74
DF2 gDNA 4-5-12 DF2 AmpR6.3.Ind75
H0 gDNA 4-5-12 H0 AmpR6.3.Ind76
H7 gDNA 4-5-12 H7 AmpR6.3.Ind77
H14 gDNA 4-5-12 H14 AmpR6.3.Ind78
H16 gDNA 4-5-12 H16 AmpR6.3.Ind79
GM:H0:: 1:1 GM:H0 1:1 AmpR6.3.Ind80