Noi/NOTES/2012-8-23

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Bisulfite conversion[edit]

Lab IDs Conc. (ng/ul) Volume for 2000ng LabID ID H2O Total amount (ng) CT conversion reagent (ul) Total volume (ul)
GL-1 295.8 6.76 GL-1 13.24 2000.00 130.00 150.00
GL-2 220.6 9.07 GL-2 10.93 2000.00 130.00 150.00
GL-3 149.7 13.36 GL-3 6.64 2000.00 130.00 150.00
GL-4 172.3 11.61 GL-4 8.39 2000.00 130.00 150.00
GL-5 285.2 7.01 GL-5 12.99 2000.00 130.00 150.00
GL-6 296.6 6.74 GL-6 13.26 2000.00 130.00 150.00
GL-7 320.0 6.25 GL-7 13.75 2000.00 130.00 150.00
GL-8 339.3 5.89 GL-8 14.11 2000.00 130.00 150.00
GL-9 467.5 4.28 GL-9 15.72 2000.00 130.00 150.00
GL-10 431.7 4.63 GL-10 15.37 2000.00 130.00 150.00
GL-11 349.1 5.73 GL-11 14.27 2000.00 130.00 150.00
GL-12 234.5 8.53 GL-12 11.47 2000.00 130.00 150.00
GL-13** 72.2 20.00 GL-13 0.00 1444.80 130.00 150.00
GL-14 137.3 14.56 GL-14 5.44 2000.00 130.00 150.00
GL-15 207.5 9.64 GL-15 10.36 2000.00 130.00 150.00
GL-16 135.5 14.76 GL-16 5.24 2000.00 130.00 150.00
AL-1 226.6 8.83 AL-1 11.17 2000.00 130.00 150.00
AL-2 375.5 5.33 AL-2 14.67 2000.00 130.00 150.00
AL-3 256.8 7.79 AL-3 12.21 2000.00 130.00 150.00
AL-4 142.1 14.08 AL-4 5.92 2000.00 130.00 150.00
AL-5 140.1 14.27 AL-5 5.73 2000.00 130.00 150.00
  • Note: Sample GL-5, GL-7 and GL-8 have lower yield than other samples, so I did bis-cvt in duplicate
Program
1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
  • Elute with 30ul Elution buffer, after Qubit quantification I have ~25ul for BSPP capture

330k BSPP capture set up[edit]

Probe calculation[edit]

Subset ID Adjusted Multiplier(2012_05_22) Actual amount of probe required (probe:target ratio, 100:1) Multiplier F Probe concentration (ng/ul) Volume (ul) Volume for 23rxn (ul) Subset ID
DMR.s1 0.25 2.24 0.56 26.2 0.02 0.49 DMR.s1
DMR.s2 0.35 2.24 0.78 30.1 0.03 0.60 DMR.s2
DMR.s3 1.15 18.90 21.74 21.15 1.03 23.64 DMR.s3
DMR.s4 1.00 18.90 18.90 22.34 0.85 19.46 DMR.s4
DMR.s5 4.83 18.90 91.29 29 3.15 72.40 DMR.s5
DMR.Exp1 1.00 18.90 18.90 31.88 0.59 13.64 DMR.Exp1
DMR.Exp2 1.00 18.90 18.90 24.11 0.78 18.03 DMR.Exp2
DMR.Exp3 1.00 18.90 18.90 28.9 0.65 15.04 DMR.Exp3
1.05uM Oligo suppressor-set1 0.125 2.88 1.05uM Oligo suppressor-set1
1.05uM Oligo suppressor-set2 0.125 2.88 1.05uM Oligo suppressor-set2
  • This means that the maximum volume of bis-cvt gDNA is 42.15ul in total volume 55ul

Reaction mix[edit]

Components 1rxn (ul) 23 rxn mix (ul)
10X AmpLigase Buffer 5.50 126.50
DMR330k probe mix (ul) 7.10 163.30
105nM Oligo suppressor 0.25 5.75
Total volume (ul) 12.85 295.55


Sample IDs Conc. (ng/ul) Volume for 250ng (ul) Sample IDs H2O Total amount of
bis-cvt gDNA (ng)
Probe mix (ul) 10X AmpLigase Buffer 1.05uM Oligo suppressor
set1 &2 (ul)
Total volume (ul)
GL-1 6.30 39.68 GL-1 2.47 250.00 7.10 5.50 0.25 55.00
GL-2 5.00 42.15 GL-2 0.00 210.75 7.10 5.50 0.25 55.00
GL-3 8.92 28.03 GL-3 14.12 250.00 7.10 5.50 0.25 55.00
GL-4 7.14 35.01 GL-4 7.14 250.00 7.10 5.50 0.25 55.00
GL-5* 5.34 42.15 GL-5* 0.00 225.08 7.10 5.50 0.25 55.00
GL-6## 3.97 42.15 GL-6## 0.00 167.34 7.10 5.50 0.25 55.00
GL-7* 5.30 42.15 GL-7* 0.00 223.40 7.10 5.50 0.25 55.00
GL-8* 5.08 42.15 GL-8* 0.00 214.12 7.10 5.50 0.25 55.00
GL-9 5.51 42.15 GL-9 0.00 232.25 7.10 5.50 0.25 55.00
GL-10 5.31 42.15 GL-10 0.00 223.82 7.10 5.50 0.25 55.00
GL-11 4.99 42.15 GL-11 0.00 210.33 7.10 5.50 0.25 55.00
GL-12 4.97 42.15 GL-12 0.00 209.49 7.10 5.50 0.25 55.00
GL-13 7.11 35.16 GL-13 6.99 250.00 7.10 5.50 0.25 55.00
GL-14 7.27 34.39 GL-14 7.76 250.00 7.10 5.50 0.25 55.00
GL-15 8.01 31.21 GL-15 10.94 250.00 7.10 5.50 0.25 55.00
GL-16 7.25 34.48 GL-16 7.67 250.00 7.10 5.50 0.25 55.00
AL-1 5.06 42.15 AL-1 0.00 213.28 7.10 5.50 0.25 55.00
AL-2 5.00 42.15 AL-2 0.00 210.75 7.10 5.50 0.25 55.00
AL-3 5.22 42.15 AL-3 0.00 220.02 7.10 5.50 0.25 55.00
AL-4 7.16 34.92 AL-4 7.23 250.00 7.10 5.50 0.25 55.00
AL-5 8.40 29.76 AL-5 12.39 250.00 7.10 5.50 0.25 55.00
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (08.24.2012, ~5pm)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

SLN mix solution[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume72ul
AmpliTaq Stoffel 10 U/ul 2 U/ul 14.40
AmpLigase 5 U/ul 0.5 U/ul 7.20
dNTP 1000 uM 100 uM 7.20
10x AmpLigase Buffer 10 x 1 x 7.20
H2O 36.00
Total 72.00
  • Add 3ul of SLN mix to each

2012.08.25

PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 Ind9-29)[edit]

  • I tested if the capture works well by performing qPCR of few samples in small volume 25ul
  • Sample te be tested: NTC, GL7,GL8 AL-1 and AL3
  • Since the total volume of capture reaction is large (55ul), I will add more template in the PCR reaction to the previous experiment (~20% V/V -> 5ul in 25ul reaction)
Components 1 rxn 5.2x rxn mix
Circularized DNA template 5.00 0.00
10uM AmpF6.4Sol 0.50 2.60
10uM AmpR6.3Sol 0.50 2.60
50x SYBG 0.20 1.04
2x Phusion MM 12.50 65.00
H2O 6.30 32.76
Total 25.00 104.00
  • Aliquot 20ul, add 5ul of DNA template
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
File:20120825 testqPCR BSPP-GL-AL.png
Components 1 rxn 45x rxn mix
Captured template 20.00 0.00
10uM AmpF6.4Sol 2.00 90.00
10uM AmpR6.3Indx (X=9-29) 2.00 0.00
50x SYBG 0.80 36.00
2x Phusion MM 50.00 2250.00
H2O 25.20 1134.00
Total 100.00 3510.00

Program

98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x11 -> 72C 3min
(All reactions were stopped at cycle 19)
File:20120825 qPCR BSPP-GL-AL.png
Sample IDs Indx Sample IDs Indx Sample IDs Indx
GL-1 AmpR6.3Indx9 GL-9 AmpR6.3Indx17 AL-1 AmpR6.3Indx25
GL-2 AmpR6.3Indx10 GL-10 AmpR6.3Indx18 AL-2 AmpR6.3Indx26
GL-3 AmpR6.3Indx11 GL-11 AmpR6.3Indx19 AL-3 AmpR6.3Indx27
GL-4 AmpR6.3Indx12 GL-12 AmpR6.3Indx20 AL-4 AmpR6.3Indx28
GL-5 AmpR6.3Indx13 GL-13 AmpR6.3Indx21 AL-5 AmpR6.3Indx29
GL-6 AmpR6.3Indx14 GL-14 AmpR6.3Indx22
GL-7 AmpR6.3Indx15 GL-15 AmpR6.3Indx23
GL-8 AmpR6.3Indx16 GL-16 AmpR6.3Indx24
- Took 80ul of PCR product from replicate1 and 2 (total volume 160ul)
- Purified with 0.8 vol of AmPure beads (I diluted AMPure bead 1:1 ratio with 20%PEG/2.5M NaCl)
- Eluted with 60ul EB buffer
- Performed PAGE quantification by loading sample 1ul
File:ZhangLab 2 2012-08-25 21hr 25min PQ GL-1.jpg  File:ZhangLab 2 2012-08-25 21hr 25min PQ GL-2.jpg
Sample IDs Conc. (ng/ul) Yields in 60ul (ng) Volume for 200ng (ul)
GL-1 10.72 642.91 18.67
GL-2 12.24 734.53 16.34
GL-3 11.98 718.56 16.70
GL-4 12.17 730.11 16.44
GL-5 11.07 664.39 18.06
GL-6 12.74 764.43 15.70
GL-7 10.40 623.89 19.23
GL-8 9.74 584.46 20.53
GL-9 10.88 652.62 18.39
GL-10 11.16 669.83 17.92
GL-11 8.30 497.79 24.11
GL-12 9.40 564.06 21.27
GL-13 9.26 555.74 21.59
GL-14 7.18 430.56 27.87
GL-15 7.37 441.94 27.15
GL-16 8.25 494.83 24.25
AL-1 5.11 306.64 39.13
AL-2 6.38 382.88 31.34
AL-3 5.96 357.37 33.58
AL-4 4.91 294.31 40.77
AL-5 4.45 266.78 44.98
- Combine 200ng of each library.
-Perform PAGE size selection in 6% TBE 5-well gel (3 gels)
File:ZhangLab 2 2012-08-26 18hr 46min PAGE SS-1.jpg File:ZhangLab 2 2012-08-26 18hr 29min PAGE SS-2.jpg
* From gel images, DNA was overoaded
- Resuspended with H2O, total volume 48ul

File:ZhangLab 2 2012-08-26 22hr 24min PQ NP BSPP GL-AL.Ind9-29.jpg

dsDNA Qubit HS quantification[edit]

Library IDs Concentration in the Qubit Unit uL used Dilution Final conc. (ng/ul) Ave. Conc. (ng/ul)
AL-exome_Aug21 1ul 46.60 ng/mL 1.00 200 9.32 9.76
AL-exome_Aug21 2ul 102.00 ng/mL 2.00 100 10.20
NP_WGBS_N37_Aug26 1ul 18.70 ng/mL 1.00 200 3.74 3.80
NP_WGBS_N37_Aug26 2ul 38.50 ng/mL 2.00 100 3.85
NP-BSPP-GL-AL_Ind9-29_Aug23 1ul 72.50 ng/mL 1.00 200 14.50 14.95
NP-BSPP-GL-AL_Ind9-29_Aug23 2ul 154.00 ng/mL 2.00 100 15.40


Library IDs Ave. Conc. (ng/ul) Size (bp) Conc. (nM) MW (g/mole)
AL-exome_Aug21 9.76 300 53.52 182,377.9
NP_WGBS_N37_Aug26 3.80 500 12.49 303,857.9
NP-BSPP-GL-AL_Ind9-29_Aug23 14.95 375 65.59 227,932.9
- Quantified concentration by Qubit dsDNA assay HS (based on size 375bp): 14.95ng/ul or 65.59nM
- Concentration by PAGE quantification is 26.88ng/ul
  • Library IDs: NP-BSPP-GL-AL_Ind9-29_Aug23, 2012, sequenced on HiSeqAug28 run at Biogem (Lane : 7-8)