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 0.7vol. Ampure beads, elute with 25ul EB/tube, so total volume = 75ul
- PAGE quantify DNA concentration (loaded sample 1ul)

File:ZhangLab 2 2011-02-22 15hr 46min 1st Amp P1C-P8C.jpg File:ZhangLab 2 2011-02-22 15hr 46min 1st Amp P1P-P2P2.jpg

PAGE quantification and MmeI digestion set up

Sample ID Conc.(ng/ul) Total volume(ul) Total amount (ng) 10X NEB buffer4 32mM SAM MmeI Total volume(ul)
P1C 2.09 75.00 156.39 9.30 0.30 8.00 92.60
P2C 4.67 75.00 350.41 9.30 0.30 8.00 92.60
P3C 3.09 75.00 231.84 9.30 0.30 8.00 92.60
P4C 4.24 75.00 317.74 9.30 0.30 8.00 92.60
P5C 3.45 75.00 258.94 9.30 0.30 8.00 92.60
P6C 4.19 75.00 314.13 9.30 0.30 8.00 92.60
P7C1 3.74 75.00 280.28 9.30 0.30 8.00 92.60
P7C2 2.14 75.00 160.76 9.30 0.30 8.00 92.60
P8C 3.21 75.00 240.62 9.30 0.30 8.00 92.60
P1P 3.35 75.00 251.00 9.30 0.30 8.00 92.60
P2P 3.18 75.00 238.35 9.30 0.30 8.00 92.60
P3P 3.01 75.00 225.81 9.30 0.30 8.00 92.60
P4P 5.10 75.00 382.61 9.30 0.30 8.00 92.60
P5P 4.06 75.00 304.36 9.30 0.30 8.00 92.60
P6P 3.22 75.00 241.75 9.30 0.30 8.00 92.60
P7P 3.68 75.00 276.35 9.30 0.30 8.00 92.60
P8P1 4.03 75.00 302.36 9.30 0.30 8.00 92.60
P8P2 7.34 75.00 550.50 9.30 0.30 8.00 92.60


Reagents 1x 18.5 rxn mix
10X NEB buffer4 9.30 172.05
32mM SAM 0.30 5.55
MmeI 8.00 148.00
Total 17.60 aliquot 17.6ul, add 75ul of amplified amplicon


  • incubate 37C 2hr
  • purify with 0.7vol. Ampure beads, elute with 30ul EB
  • PAGE quantify DNA concentration (loaded sample 2ul)

File:ZhangLab 2 2011-02-22 20hr 38min MmeI digestion P1C-P8C.jpg File:ZhangLab 2 2011-02-22 20hr 41min MmeI digestion P1P-P8P2.jpg