Dinh/NOTES/2010-10-26

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Materials[edit]

Reagents and Kits Source Cat#
EZ DNA Methylation Gold Kit Zymo Research D5005
10x Ampligase buffer Epicentre A1905B
Ampligase Epicentre A3210K
dNTP New England Biolab N0447L
Stoffel fragment Applied Biosystems N808-0038
ExoI Epicentre X40520K
ExoIII Epicentre EX4425K
2xPhusion HF Master Mix New England Biolab F-531L
MmeI New England Biolab R0637L
Quick Ligation Kit New England Biolab M2200S
SYBR GreenI nucleic acid gel stain Invitrogen S7585
Agencourt AMPure XP Beckman Coulter A63880
100% Ethanol
QIAQuick PCR Purification Kit Qiagen 28106
E-Gel Size Select 2% Invitrogen G6610-02


Materials Source Cat#
Low Tube Strips, CLR BioRad TLS0801
Microseal 'B' Film BioRad MSB1001
Barrier Tips, or low retention pipette tips Neptune BT200, BT10E, BT1000, BT20
Mineral Oil Sigma M5904-500ml
6% TBE Gel 1.0mmx10well Invitrogen EC6265BOX
10x TBE buffer for PAGE National Diagnostics EC-860
SYBR Gold nucleic acid gel stain Invitrogen S11494


Primers Sequence Modifications
AmpF6.3NH2 /5AmMC6/CAGATGTTATCGAGGTCCGAC 5' Amino modifier C6
AmpR6.3NH2 /5AmMC6/GGAACGATGAGCCTCCAAC 5' Amino modifier C6
PE_t_N2, Y-adapter for MmeI ligation-top ACACTCTTTCCCTACACGACGCTCTTCCGATCTN*N 3'-Phosphorothioate bond
PE_b_A, Y-adapter for MmeI ligation-bottom /5Phos/AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5'-Phosphorylation
PCR_Fs AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTC None
PCR_R.N2Ind1 CAAGCAGAAGACGGCATACGAGATCGTGATCTCGGCATTCCTGCTGAACCGCTCTT None
PCR_R.N2Ind2 CAAGCAGAAGACGGCATACGAGATACATCGCTCGGCATTCCTGCTGAACCGCTCTT None
PCR_R.N2Ind3 CAAGCAGAAGACGGCATACGAGATGCCTAACTCGGCATTCCTGCTGAACCGCTCTT None
PCR_R.N2Ind4 CAAGCAGAAGACGGCATACGAGATTGGTCACTCGGCATTCCTGCTGAACCGCTCTT None

Bisulfite Conversion[edit]

Perform bisulfite conversion according to instructions given in EZ Methylation Gold Kit.

DMR220K Normalization Capture[edit]

  • gDNA template: bis-cvt BJ-iPS (145.5ng/ul, Nanodrop)

Calculations[edit]

Probe/Target Ratio 100 100 100
Probes size 6500 27000 55000
Template (ng) 200 200 200
Human gDNA MW (g/mole) 1.82E+012 1.82E+012 1.82E+012
Human gDNA(moles) 1.10E-019 1.10E-019 1.10E-019
Probes required (moles) 1.10E-017 1.10E-017 1.10E-017
Probes MW (g/mole) 2.03E+008 8.45E+008 1.72E+009
Amount probes (ng) 2.23 9.27 18.88

Probes Subset Information[edit]

  • Probes were prepared by DD and ZL.
  • We re-quantified the probes and got higher concentration, the issue was probably not shaking in SYBR Gold solution long enough.

Experimental Setup[edit]

  • We prepared the reactions in PCR tubes
  • Buffer used is 10x Ampligase Buffer
  • Set up capture reaction in low profile strip tubes (or PCR tubes/plates).
  • Spin down the reaction mix and add 1-2 droplets of mineral oil with P200.
  • Use Microseal film to cover the tubes.
  • First 7 reactions: Capture with individual probe set.
Amount Probes DMR subset Probes(ul) Template(ul) Buffer(ul) H2O(ul) Total(ul)
2.23ng DMR.s1 11.08 1.37 2 5.54 20
2.23ng DMR.s2 8.81 1.37 2 7.81 20
18.88 DMR.s3 0.5 1.37 2 16.12 20
18.88 DMR.s4 3.59 1.37 2 13.04 20
18.88 DMR.s5 3.05 1.37 2 13.58 20
9.27 DMR.s6 1.34 1.37 2 15.28 20
9.27 DMR.s7 2.09 1.37 2 14.53 20

Program

   95c 30sec -> cool down to 58C at 0.02C/sec -> 58C 20h 
   -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) 
   -> 58C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 1h -> 94C 2min -> 4C hold.
Adding 2ul SLN mix after >20 h of 58C incubation:
Leave tubes in thermocycler and just remove Microseal film cover to add SLN mix.
Use P10, low retention tips and make sure to reach the bottom of the tube before dispensing.
Dispense slowly, then slowly pull out the pipette tip before releasing hold on dispenser.
Ensure that all reagents were pumped out. 
Cut new Microseal films to cover the tubes.
Adding 2ul ExoI/III mix after 3-4 h of 58C incubation:
Perform same steps as adding SLN. 
SLN Mix 1x 16x Final Conc
H2O 0.55 8.8
dNTP(1mM) 0.05 0.8 50uM
10xAmpligase Buffer 0.1 1.6 1x
AmpLigase 0.1 1.6 0.5U/ul
Stoffel 0.2 3.2 2U/ul
TOTAL 1 16

For ExoI/III mix, add equal volumes of Exo I and Exo III.

RT PCR[edit]

Reagent Concentration Vol (ul) Final Concentration 11x Master Mix
Template --- 10
AmpR6.3NH2 10uM 2 0.2uM 22
AmpF6.3NH2 10uM 2 0.2uM
Phusion HF 2x MM 2x 50 1x 550
SYBR Green 50x 0.4 1x 4.4
H2O --- 36 396
TOTAL 100.4 972.4

Program

98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x15 ->72C 3min 
Purified each reaction with 0.7x AMPure Beads.
File:ZhangLab 2 2010-10-07 DMR220KNorm.jpg
1 ul of each subset loaded.
We combined s1-s7, A, and B to form the barcoded DMR library, then performed PAGE purification.

Solexa sequencing library construction[edit]

  1. MmeI digestion
  2. Ligation of PE_N2 adapter
  3. PCR with barcoded primers
  4. Combine libraries and perform PAGE purification
  5. Quantification by qPCR