Noi/NOTES/2011-3-18
- Link to calendar: [[1]]
DMR220k capture (Probe set S1-S3 plus Exp1-Exp3) and library construction of cell samples from Belmonte's lab[edit]
- continued form 2011_03_10: [[2]]
Determine bisulfite converted gDNA concentration using Qubit Fluorometer[edit]
Sample preparation
- Add 1ul of BIS-CVT gDNA to 199ul of Quant-iT working solution
Sample | Concentration in the Qubit | Unit | uL used | Dilution | Sample Concentration | Unit | Final conc. (ng/ul) | Yields (ng) in 15ul |
1. HGPS | 75.80 | ng/mL | 1.00 | 200.00 | 75.80 | ng/mL | 15.16 | 227.40 |
2. cHGPS1 | 90.30 | ng/mL | 1.00 | 200.00 | 90.30 | ng/mL | 18.06 | 270.90 |
3. cHGPS40 | 68.50 | ng/mL | 1.00 | 200.00 | 68.50 | ng/mL | 13.70 | 205.50 |
4. AWS | 55.30 | ng/mL | 1.00 | 200.00 | 55.30 | ng/mL | 11.06 | 165.90 |
5. cAWS1 | 66.10 | ng/mL | 1.00 | 200.00 | 66.10 | ng/mL | 13.22 | 198.30 |
Capture set up[edit]
Probe calculation[edit]
- From Parkinson's patient sample sequencing, Dr. Zhang suggested me to adjust the ratio of each subset of the probe. In this study and future study, I will use DMR 220k set S1-S3 (keep the same captured efficiency coefficient) and Exp1-3 by using the same ratio as S2 set. Then set S2 or S3 set as the baseline. In this experiment I set S2 as the baseline.
- Probe :target ration = 100:1
- gDNA template amount 200ng
Probes | Size | Amount of probe requited (ng) |
Multiplier D | Actual amount of probe required (ng) amount of probe required x Multiplier D |
Conc. (ng/ul) | Probe volume (ul) | 6.2 rxn mix |
DMR220k.S1 | 6500.00 | 2.23 | 0.56 | 1.25 | 2.00 | 0.62 | 3.87 |
DMR220k.S2 | 6500.00 | 2.23 | 1.00 | 2.23 | 2.00 | 1.12 | 6.91 |
DMR220k.S3 | 55000.00 | 18.90 | 2.78 | 52.54 | 23.20 | 2.26 | 14.04 |
DMR220k.Exp1 | 55000.00 | 18.90 | 1.00 | 18.90 | 33.00 | 0.57 | 3.55 |
DMR220k.Exp2 | 55000.00 | 18.90 | 1.00 | 18.90 | 25.00 | 0.76 | 4.69 |
DMR220k.Exp3 | 55000.00 | 18.90 | 1.00 | 18.90 | 29.00 | 0.65 | 4.04 |
Total volume | 5.98 | 37.10 |
Sample ID | Sample code | Conc. (ng/ul) | Volume (ul) | H2O | Total amount of bis-cvt gDNA | probe mix (ul) | 10x AmpLigase buffer (ul) | 105nM Oligo suppressor (ul) | H20 | Total volume |
1. HGPS | BM1 | 15.16 | 13.50 | 0.00 | 204.66 | 5.98 | 2.30 | 1.00 | 0.22 | 23.00 |
2. cHGPS1 | BM2 | 18.06 | 11.00 | 2.50 | 198.66 | 5.98 | 2.30 | 1.00 | 0.22 | 23.00 |
3. cHGPS40 | BM3 | 13.70 | 13.50 | 0.00 | 184.95 | 5.98 | 2.30 | 1.00 | 0.22 | 23.00 |
4. AWS | BM4 | 11.06 | 13.50 | 0.00 | 149.31 | 5.98 | 2.30 | 1.00 | 0.22 | 23.00 |
5. cAWS1 | BM5 | 13.22 | 13.50 | 0.00 | 178.47 | 5.98 | 2.30 | 1.00 | 0.22 | 23.00 |
6. NTC | NTC | NA | 0.00 | 13.50 | 0.00 | 5.98 | 2.30 | 1.00 | 0.22 | 23.00 |
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) (2011_03_19) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
SLN preparation[edit]
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume (17ul) |
AmpliTaq Stoffel | 10.00 | U/ul | 2.00 | U/ul | 3.40 |
AmpLigase | 5.00 | U/ul | 0.50 | U/ul | 1.70 |
dNTP | 1.00 | mM | 100.00 | uM | 1.70 |
10x AmpLigase Buffer | 10.00 | x | 1.00 | x | 1.70 |
H2O | 8.50 | ||||
aliquot 2.5ul to each tube |
PCR Amplification[edit]
(2011_03_21)
- amplify 2tubes/ sample
- 5samples x 2 = 10 plus 1 NTC = 11rxn
Reagents | volume (ul) | 11.5 rxn mix |
captured template | 9.00 | 0.00 |
100uM AmpFV6.3NH2 | 0.20 | 2.30 |
100uM AmpRV6.3NH2 | 0.20 | 2.30 |
50x SYBG | 0.40 | 4.60 |
2x PhusionHF MM | 50.00 | 575.00 |
H2O | 40.20 | 462.30 |
Total volume (ul) | 100.00 | aliquot 91ul, add 9ul of captured DNA |
Program
98C 30sec (98C 10sec -> 58C 20sec-> 72C 20sec) x 20 cycles 72C 2min 15C hold.
File:2011 03 21 AmpFR63NH2 rawdata.PNG
Ct value
- HGPS : 13.89
- cHGPS1: 12.87
- cHGPS40: 13.02
- AWS: 12.84
- cAWS1: 16.59
- purify with 0.7vol. Ampure beads, elute with 25ul EB/tube, so total volume = 50ul
- PAGE quantify DNA concentration (loaded sample 1ul)
File:ZhangLab 2 2011-03-21 21hr 54min AmpFR63NH2 1ul.jpg
PAGE quantification and MmeI digestion set up[edit]
Sample ID | Conc.(ng/ul) | Total volume(ul) | Total amount (ng) | 10X NEB buffer4 | 32mM SAM | MmeI | Total volume(ul) |
1. HGPS | 8.71 | 48.00 | 417.96 | 6.00 | 0.19 | 6.00 | 60.19 |
2. cHGPS1 | 8.84 | 48.00 | 424.30 | 6.00 | 0.19 | 6.00 | 60.19 |
3. cHGPS40 | 9.20 | 48.00 | 441.75 | 6.00 | 0.19 | 6.00 | 60.19 |
4. AWS | 11.60 | 48.00 | 556.63 | 6.00 | 0.19 | 6.00 | 60.19 |
5. cAWS1 | 9.95 | 48.00 | 477.61 | 6.00 | 0.19 | 6.00 | 60.19 |
Reagents | 1x | 5.5 rxn mix |
10X NEB buffer4 | 6.00 | 33.00 |
32mM SAM | 0.19 | 1.05 |
MmeI | 6.00 | 33.00 |
Total | 12.19 | aliquot 12.19ul, add 48ul of amplified amplicon |
- incubate 37C 1hr
- purify with 0.7vol. Ampure beads, elute with 30ul EB
- PAGE quantify DNA concentration (loaded sample 2ul)
File:ZhangLab 2 2011-03-22 00hr 48min MmeI digestion.jpg
Sample ID | Conc of MmeI completely digested DNA (ng/ul) | Total volume (ul) | Total amount (ng) |
1. HGPS | 4.27 | 30.00 | 128.05 |
2. cHGPS1 | 5.46 | 30.00 | 163.91 |
3. cHGPS40 | 4.95 | 30.00 | 148.48 |
4. AWS | 5.55 | 30.00 | 166.59 |
5. cAWS1 | 5.28 | 30.00 | 158.29 |
N2 adaptor ligation set up[edit]
Adaptor preparation[edit]
Components | Volume (ul) | Final conc. (uM) |
100uM PE_N2_adaptor | 2.00 | 20.00 |
100uM PE_b_A | 2.00 | 20.00 |
10x Stoffel buffer | 1.00 | 1x |
H2O | 5.00 | |
Total | 10.00 |
Program
94C 2min -> 0.2C/sec to 20C -> 4C hold
Adapters to ligated product ratio: 10:1
Est length of digested products: 212bp (after MmeI)
MW digested products = (212bp*607.4 D/bp +157.9 D) = 128.927kD = 128,927 g/mole
For 100 ng digested product = 100ng / 128,927g/mole = 7.76E-4 nmole * 10:1 = 0.008 nmole adapters required.
20uM Adapters (ul) = 0.008nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.008nmoles/ (20E-3 nmoles/ul) = 0.40 ul
- 0.40 ul of 20uM adapters per 100ng digested products
Sample ID | conc.(ng/ul) | Volume (ul) for 100ng | 20uM N2 adaptors volume (ul) | 5xQuickLigase buffer | QuickLigase (ul) | H2O (ul) | Total (ul) |
1. HGPS | 4.27 | 23.43 | 0.40 | 8.00 | 2.00 | 6.17 | 40.00 |
2. cHGPS1 | 5.46 | 18.30 | 0.40 | 8.00 | 2.00 | 11.30 | 40.00 |
3. cHGPS40 | 4.95 | 20.21 | 0.40 | 8.00 | 2.00 | 9.39 | 40.00 |
4. AWS | 5.55 | 18.01 | 0.40 | 8.00 | 2.00 | 11.59 | 40.00 |
5. cAWS1 | 5.28 | 18.95 | 0.40 | 8.00 | 2.00 | 10.65 | 40.00 |
6. NTC | NA | 0.00 | 0.40 | 8.00 | 2.00 | 29.60 | 40.00 |
Reagents | 1x | 6.5 rxn mix |
5xQuickLigase buffer | 8.00 | 52.00 |
QuickLigase | 2.00 | 13.00 |
N2 Adapters, conc. (Gr. A, B or C) | 0.40 | 2.60 |
Total | 10.40 | aliquot 10.4ul, add MmeI digested DNA, adjust volume with H2O |
- incubate at RT for 15 min
- purify with 1 vol. Ampure beads, elute with 30ul EB
Amplification[edit]
- Use 1% N2 ligated DNA template for amplification
- 2tubes/sample: 5x2 = 10rxn plus 1NTC = 11rxn
Reagents | 1 rxn | 11.5rxn mix |
N2 adapter ligated template | 1.00 | 0.00 |
10uM PCR_F | 1.00 | 11.50 |
10uM PCR_R.N2 IndX(X= 8,9,10,…,12) | 1.00 | 0.00 |
50x SYBG | 0.40 | 4.60 |
2x PhusionHF MM | 50.00 | 575.00 |
H2O | 46.60 | 535.90 |
Total volume (ul) | 100.00 | aliquot 98ul, add 1ul template, 1ul PCR_R.N2 IndX |
Sample IDs | IndX |
1. HGPS | PCR_R.N2Ind8 |
2. cHGPS1 | PCR_R.N2Ind9 |
3. cHGPS40 | PCR_R.N2Ind10 |
4. AWS | PCR_R.N2Ind11 |
5. cAWS1 | PCR_R.N2Ind12 |
6. NTC | PCR_R.N2Ind12 |
Program
98C 30sec (98C 10sec -> 64C 20sec-> 72C 30sec) x 11 cycles 72C 2min 15C hold.
File:2011 03 21 N2Amp rawdata.PNG
- Purify amplified amplicon with 0.7volume Ampure beads, elute with 50ul EB total
- PAGE quantify DNA concentration (loaded sample 1ul)
File:ZhangLab 2 2011-03-22 05hr 04min PQ SeqLib (1).jpg
Sample IDs | IndX | Conc. (ng/ul) | Total volume (ul) | Total amount (ng) | Volume for 65ng (ul) |
1. HGPS | PCR_R.N2Ind8 | 1.59 | 50.00 | 79.36 | 40.95 |
2. cHGPS1 | PCR_R.N2Ind9 | 1.89 | 50.00 | 94.67 | 34.33 |
3. cHGPS40 | PCR_R.N2Ind10 | 1.74 | 50.00 | 87.08 | 37.32 |
4. AWS | PCR_R.N2Ind11 | 2.00 | 50.00 | 100.06 | 32.48 |
5. cAWS1 | PCR_R.N2Ind12 | 1.35 | 50.00 | 67.33 | 48.27 |
- perform PAGE size selection with 6% TBE 5-well gel (1gels)
File:ZhangLab 2 2011-03-22 06hr 24min PAGE-SS.jpg
- Total volume of of sequencing libraries: 30ul
File:ZhangLab 2 2011-03-22 05hr 04min PAGE-SS SeqLib-HL091.jpg
- 0.6 and 0.3ul of sequencing libraries were analyzed by PAGE quantification, conc.: 4.62ng/ul, 22.6nM (336bp), yields = 4.62ng/ul * 30ul = 138ng
- Sequencing: Sample ID: NP-220K-Belmonte_Ind8-12-March10, HL091 run
- In the same gel, ALice's samples were included (loaded sample 1ul)
File:ZhangLab 2 2011-03-22 16hr 13min PQ SeqLib PAGE-SS AL.jpg
- AL1: conc. = 61.03ng/ul
- AL4: conc. = 51.39ng/ul
- AL6: conc. = 68.47ng/ul
- AL8: conc. = 12.52ng/ul
- Note: Since sample cAWS1 had the captured PCR curve with high Ct value (similar to some sample from PD samples which had low quality), so Dr. Zhang concerns about the quality of this sample. However he suggested me to move on sequencing, and in the next batch of sample include cAWS1 and use 4x more bisulfite converted DNA (but do the bisulfite conversion with 1ug of gDNA per column).