Noi/NOTES/2011-2-15
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DMR220k capture(Probe set S1-S5 plus new probe set Exp1-Exp3) and library construction of Parkinson's patients
- Received 18 human brain samples from Pakinson's patients. This includes control and Pakinson's patiants (2011_02_02)
- Human frozen cerebral cortex
Pair | Control Sample ID | Sample code | Sex | age (year.day) | Postmortem time | Conc. (ug/ul)/ volume (ul) | PD Sample ID | Sample code | Sex |
1. | 5028 | P1C | M | 67.29 | P18 | 0.5ug/ul/ 4ul | 1947 | P1P | M |
2. | 4789 | P2C | F | 72.05 | P19 | 0.5ug/ul/ 4ul | 4977 | P2P | F |
3. | 5171 | P3C | M | 79.09 | P05 | 0.5ug/ul/ 4ul | 4879 | P3P | M |
4. | 1818 | P4C | M | 76.29 | P03 | 0.5ug/ul/ 4ul | 4526 | P4P | M |
5. | 5089 | P5C | M | 89.02 | P14 | 0.5ug/ul/ 4ul | 5203 | P5P | M |
6. | 5237 | P6C | M | 52.29 | P13 | 0.5ug/ul/ 4ul | 1910 | P6P | M |
7. | 1569 | P7C1 | F | 77.09 | P08 | 0.5ug/ul/ 4ul | 1401 | P7P | F |
7. | 5219 | P7C2 | F | 76.35 | P03 | 0.5ug/ul/ 4ul | |||
8. | 4735 | P8C | M | 73.18 | P21 | 0.5ug/ul/ 4ul | 1741 | P8P1 | M |
8. | 5306 | P8P2 | M |
Bisulfite conversion
- gDNA total volume 4ul (0.5ng/ul) so total amount = 2ug
- Add EB 6 ul to make volume to 10ul, so the final concentration should be 200ng/ul
Sample ID | Conc.(ng/ul) | Volume (ul) | H20 (ul) | CT conversion reagent (ul) | Total volume (ul) |
P1C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P2C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P3C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P4C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P5C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P6C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P7C1 | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P7C2 | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P8C | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P1P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P2P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P3P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P4P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P5P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P6P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P7P | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P8P1 | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
P8P2 | 200.00 | 5.00 | 15.00 | 130.00 | 150.00 |
- follow standard protocol for bisulfite conversion of ZymoResearch
- elute with 17ul Elution buffer
Determine bisulfite converted gDNA concentration using Qubit Fluorometer
Sample preparation
- Add 1ul of BIS-CVT gDNA to 199ul of Quant-iT working solution
Sample ID | Concentration in the Qubit | ' | uL used | Dilution | Sample Concentration | ' | Final conc.(ng/ul) | Yields (ng) |
P1C | 150.00 | ng/mL | 1.00 | 200.00 | 150.00 | ng/mL | 30.00 | 510.00 |
P2C | 118.00 | ng/mL | 1.00 | 200.00 | 118.00 | ng/mL | 23.60 | 401.20 |
P3C | 59.40 | ng/mL | 1.00 | 200.00 | 59.40 | ng/mL | 11.88 | 201.96 |
P4C | 321.00 | ng/mL | 1.00 | 200.00 | 321.00 | ng/mL | 64.20 | 1091.40 |
P5C | 34.70 | ng/mL | 1.00 | 200.00 | 34.70 | ng/mL | 6.94 | 117.98 |
P6C | 29.10 | ng/mL | 1.00 | 200.00 | 29.10 | ng/mL | 5.82 | 98.94 |
P7C1 | 33.70 | ng/mL | 1.00 | 200.00 | 33.70 | ng/mL | 6.74 | 114.58 |
P7C2 | 29.60 | ng/mL | 1.00 | 200.00 | 29.60 | ng/mL | 5.92 | 100.64 |
P8C | 57.60 | ng/mL | 1.00 | 200.00 | 57.60 | ng/mL | 11.52 | 195.84 |
P1P | 189.00 | ng/mL | 1.00 | 200.00 | 189.00 | ng/mL | 37.80 | 642.60 |
P2P | 393.00 | ng/mL | 1.00 | 200.00 | 393.00 | ng/mL | 78.60 | 1336.20 |
P3P | 337.00 | ng/mL | 1.00 | 200.00 | 337.00 | ng/mL | 67.40 | 1145.80 |
P4P | 307.00 | ng/mL | 1.00 | 200.00 | 307.00 | ng/mL | 61.40 | 1043.80 |
P5P | 19.10 | ng/mL | 1.00 | 200.00 | 19.10 | ng/mL | 3.82 | 64.94 |
P6P | 48.00 | ng/mL | 1.00 | 200.00 | 48.00 | ng/mL | 9.60 | 163.20 |
P7P | 18.90 | ng/mL | 1.00 | 200.00 | 18.90 | ng/mL | 3.78 | 64.26 |
P8P1 | 44.60 | ng/mL | 1.00 | 200.00 | 44.60 | ng/mL | 8.92 | 151.64 |
P8P2 | 37.80 | ng/mL | 1.00 | 200.00 | 37.80 | ng/mL | 7.56 | 128.52 |
Capture set up
Probe calculation
Probe/target ratio | 100 | ' |
Probe size (sub set Exp1-3) | 55,000 each | |
Template | 200ng | |
Human gDNA MW | 1.82E+12g/mol | (3E9bp x 607.4D/bp + 157) |
Human gDNA 200ng | 1.10E-19mol | |
Probe required (100:1) | 1.10E-17 mol | |
Probe MW (each set) | 1.72E9 g/mol | (55,000 x 103bp x 303.7D/bp + 79D) |
Amount probe required | 18.90ng each |
- Note: for DMR220k subset S1-S5 used Multiplier C
Subset ID | Multiplier C | Amount probes required x multiplier (ng) | Conc(ng/ul) | Volume (ul) | 20 rxn |
DMR.s1 | 0.05 | 0.11 | 2.00 | 0.06 | 1.10 |
DMR.s2 | 0.09 | 0.21 | 2.00 | 0.11 | 2.10 |
DMR.s3 | 0.25 | 4.71 | 23.20 | 0.20 | 4.06 |
DMR.s4 | 1.00 | 18.88 | 24.80 | 0.76 | 15.23 |
DMR.s5 | 4.83 | 91.22 | 24.70 | 3.69 | 73.86 |
DMR.Exp1 | 1.00 | 18.90 | 28.00 | 0.68 | 13.50 |
DMR.Exp2 | 1.00 | 18.90 | 26.00 | 0.73 | 14.54 |
DMR.Exp3 | 1.00 | 18.90 | 25.00 | 0.76 | 15.12 |
Total volume (ul) | 6.98 | 139.51 |
- Note: used probe set DMR220k S5, Exp1-3 prepared on 2011_02_02
Reaction mix
Components | 1rxn (ul) | 20rxn mix (ul) |
10X AmpLigase Buffer | 2.55 | 51.00 |
DMR220k Exp1-3 probe mix (ul) | 6.98 | 139.60 |
105nM Oligo suppressor | 1.00 | 20.00 |
Total volume (ul) | 10.53 | 210.60 |
Sample ID | Final conc.(ng/ul) | volume for 200ng | H2O | probe mix (ul) | 10x AmpLigase buffer (ul) | 105nM Oligo suppressor (ul) | Total volume (ul) | Total amount of bis-cvt DNA captured |
P1C | 30.00 | 6.67 | 8.33 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P2C | 23.60 | 8.47 | 6.53 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P3C* | 11.88 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 178.20 |
P4C | 64.20 | 3.12 | 11.88 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P5C* | 6.94 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 104.10 |
P6C* | 5.82 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 87.30 |
P7C1* | 6.74 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 101.10 |
P7C2* | 5.92 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 88.80 |
P8C* | 11.52 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 172.80 |
P1P | 37.80 | 5.29 | 9.71 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P2P | 78.60 | 2.54 | 12.46 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P3P | 67.40 | 2.97 | 12.03 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P4P | 61.40 | 3.26 | 11.74 | 6.98 | 2.55 | 1.00 | 25.53 | 200.00 |
P5P* | 3.82 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 57.30 |
P6P* | 9.60 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 144.00 |
P7P* | 3.78 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 56.70 |
P8P1* | 8.92 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 133.80 |
P8P2* | 7.56 | 15.00 | 0.00 | 6.98 | 2.55 | 1.00 | 25.53 | 113.40 |
- Note: * less than 200ng of bis-cvt DNA in probe capture
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_02_16) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
PCR Amplification
(2011_02_17)
- amplify 2tubes/ sample
- 19samples x 2 = 38 plus 1 NTC = 39rxn
Reagents | volume (ul) | 39.5rxn mix |
captured template | 9.00 | 0.00 |
100uM AmpFV6.3NH2 | 0.20 | 7.90 |
100uM AmpRV6.3NH2 | 0.20 | 7.90 |
50x SYBG | 0.40 | 15.80 |
2x PhusionHF MM | 50.00 | 1975.00 |
H2O | 40.20 | 1587.90 |
Total volume (ul) | 100.00 | 3594.50 |
aliquot 91ul, add 9ul of captured DNA template
Program
98C 30sec (98C 10sec -> 58C 20sec-> 72C 20sec) x 20 cycles 72C 2min 15C hold. - purify with QiaQuick column (1column/sample), elute with 40ul EB - PAGE quantify DNA concentration (loaded sample 0.5ul)