Noi/NOTES/2012-8-23
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Bisulfite conversion[edit]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-8-22
- Repeat bisulfite conversion and I will fill more info and reason of repeating
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)